Alleles of the zwf gene from coryneform bacteria

ABSTRACT

The invention relates to mutants and alleles of the zwf gene of coryneform bacteria, which encode variants of the Zwf subunit of glucose 6-phosphate dehydrogenase (EC: 1.1.1.49), and to processes for preparing amino acids, in particular L-lysine and L-tryptophan, by using bacteria which harbor said alleles.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a division of U.S. application Ser. No.11/790,063, filed on Apr. 23, 2007(which issued as U.S. Pat. No.7,585,650 on Sep. 8, 2009), which is a division of Ser. No. 11/284,416,filed Nov. 22, 2005. U.S. Ser. No. 11/284,416 claims priority to Germanapplication 10 2005 013 676.1, filed on Mar 24, 2005, and the benefit ofUnited States provisional application 60/709,094, filed on Aug.18, 2005.These previous applications are incorporated by reference herein intheir entirety.

The invention relates to mutants and alleles of the zwf gene ofcoryneform bacteria, which encode variants of the Zwf subunit of glucose6-phosphate dehydrogenase (EC: 1.1.1.49), and to processes for preparingamino acids, in particular L-lysine and L-tryptophan, by using bacteriawhich harbor said alleles.

BACKGROUND OF THE INVENTION

Amino acids are applied in human medicine, in the pharmaceuticalindustry, in the food industry and especially in animal nutrition.

Amino acids are known to be prepared by fermentation of strains ofcoryneform bacteria, in particular Corynebacterium glutamicum. Due tothe great importance, continuous efforts are made to improve theproduction processes. Said processes may be improved with respect tofermentation-related measures such as, for example, stirring and oxygensupply or the composition of the nutrient media, such as, for example,sugar concentration during the fermentation, or the working-up intoproduct form, for example by means of ion exchange chromatography, orthe intrinsic performance characteristics of the microorganism itself.

The performance characteristics of said microorganisms are improved byapplying methods of mutagenesis, selection and mutant choice. Thisenables strains to be obtained which are resistant to antimetabolites orauxotrophic for metabolites which are of regulatory importance, andproduce amino acids. A known antimetabolite is the lysine analogS-(2-aminoethyl)-L-cysteine (AEC).

For some years now, methods of recombinant DNA technology have likewisebeen employed in order to improve L-amino acid-producing Corynebacteriumstrains, by amplifying individual amino acid biosynthesis genes andstudying the effect on amino acid production. A summary on a widevariety of aspects of the genetics, the metabolism and the biotechnologyof Corynebacterium glutamicum can be found in Pühler (chief ed.) inJournal of Biotechnology 104 (1-3), 1-338, 2003.

The nucleotide sequence of the gene coding for glucose 6-phosphatedehydrogenase of Corynebacterium glutamicum is generally accessible,inter alia in the database of the National Center for BiotechnologyInformation (NCBI) of the National Library of Medicine (Bethesda, Md.,USA). It can furthermore be found as sequence no. 243 (=AX065117) in thepatent application WO 01/00844.

WO 01/70995 describes an improvement in the fermentative production ofL-amino acids by coryneform bacteria due to amplification of the zwfgene.

WO 01/98472, WO 03/042389 and US 2003/0175911 A1 report novel mutationsin the zwf gene.

Moritz et al. (European Journal of Biochemistry 267, 3442-3452 (2000))report physiological and biochemical studies on glucose 6-phosphatedehydrogenase of Corynebacterium glutamicum. According to studies byMoritz et al., glucose 6-phosphate dehydrogenase consists of a Zwfsubunit and an OpcA subunit.

The microbial biosynthesis of L-amino acids in coryneform bacteria is acomplex system and linked on multiple levels to various other metabolicpathways in the cell. It is therefore not possible to predict whichmutation alters the catalytic activity of glucose 6-phosphatedehydrogenase in such a way that production of L-amino acids isimproved. It is therefore desirable to have available further variantsof glucose 6-phosphate dehydrogenase.

For reasons of better clarity, SEQ ID NO:1 depicts the nucleotidesequence of the zwf gene coding for glucose 6-phosphate dehydrogenaseand, respectively, of the zwf gene coding for the Zwf subunit of glucose6-phosphate dehydrogenase from Corynebacterium glutamicum (“wild typegene”), according to the information of the NCBI database, and SEQ IDNO:2 and 4 depict the amino acid sequence derived therefrom of theencoded glucose 6-phosphate dehydrogenase. In addition, SEQ ID NO:3indicates nucleotide sequences located upstream and downstream.

OBJECT OF THE INVENTION

The inventors have set themselves the object of providing novel measuresfor improving the production of amino acids, in particular L-lysine andL-tryptophan.

DESCRIPTION OF THE INVENTION

The invention relates to generated or isolated mutants of coryneformbacteria which preferably secrete amino acids, and which comprise a geneor allele encoding a polypeptide having glucose 6-phosphatedehydrogenase activity, wherein said polypeptide comprises an amino acidsequence in which any proteinogenic amino acid other than glycine ispresent in position 321 or a corresponding or comparable position of theamino acid sequence. Preference is given to the substitution of glycinewith L-serine.

The polypeptide which is present in the mutants of the invention maylikewise be referred to as Zwf polypeptide or Zwf subunit of glucose6-phosphate dehydrogenase.

Among the coryneform bacteria, preference is given to the genusCorynebacterium. Particular preference is given to amino acid-secretingstrains which are based on the following species:

-   -   Corynebacterium efficiens, for example the strain DSM44549,    -   Corynebacterium glutamicum, for example the strain ATCC13032,    -   Corynebacterium thermoaminogenes for example the strain FERM        BP-1539, and    -   Corynebacterium ammoniagenes, for example the strain ATCC6871,        very particular preference being given to the species        Corynebacterium glutamicum.

Some representatives of the species Corynebacterium glutamicum are alsoknown under different species names in the prior art. These include, forexample:

-   -   Corynebacterium acetoacidophilum ATCC13870,    -   Corynebacterium lilium DSM20137,    -   Corynebacterium melassecola ATCC17965,    -   Brevibacterium flavum ATCC14067,    -   Brevibacterium lactofermentum ATCC13869, and    -   Brevibacterium divaricatum ATCC14020.

Examples of known representatives of amino acid-secreting strains ofcoryneform bacteria are

the L-lysine-producing strains

-   -   Corynebacterium glutamicum DM58-1/pDM6 (=DSM4697) described in        EP 0 358 940,    -   Corynebacterium glutamicum MH20-22B (=DSM16835) described in        Menkel et al. (Applied and Environmental Microbiology 55(3),        684-688 (1989)),    -   Corynebacterium glutamicum AHP-3 (=FermBP-7382) described in EP        1 108 790,    -   Corynebacterium thermoaminogenes AJ12521 (=FERM BP-3304)        described in U.S. Pat. No. 5,250,423,

or the L-tryptophan-producing strains

-   -   Corynebacterium glutamicum K76 (=FermBP-1847) described in U.S.        Pat. No. 5,563,052,    -   Corynebacterium glutamicum BPS13 (=FermBP-1777) described in        U.S. Pat. No. 5,605,818, and    -   Corynebacterium glutamicum FermBP-3055 described in U.S. Pat.        No. 5,235,940.

Information on the taxonomic classification of strains of this group ofbacteria can be found, inter alia, in Seiler (Journal of GeneralMicrobiology 129, 1433-1477 (1983)), Kämpfer and Kroppenstedt (CanadianJournal of Microbiology 42, 989-1005 (1996)), Liebl et al.(International Journal of Systematic Bacteriology 41, 255-260 (1991))and in U.S. Pat. No. 5,250,434.

Strains denoted “ATCC” may be obtained from the American Type CultureCollection (Manassas, Va., USA). Strains denoted “DSM” may be obtainedfrom the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ,Brunswick, Germany). Strains denoted “FERM” may be obtained from theNational Institute of Advanced Industrial Science and Technology (AISTTsukuba Central 6, 1-1-1 Higashi, Tsukuba Ibaraki, Japan). TheCorynebacterium thermoaminogenes strains mentioned (FERM BP-1539, FERMBP-1540, FERM BP-1541 and FERM BP-1542) are described in U.S. Pat. No.5,250,434.

The term proteinogenic amino acids means the amino acids occurring innatural proteins, i.e. in proteins of microorganisms, plants, animalsand humans. These include in particular L-amino acids selected from thegroup consisting of L-aspartic acid, L-asparagine, L-threonine,L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine, L-cysteine,L-valine, L-methionine, L-isoleucine, L-leucine, L-tyrosine,L-phenylalanine, L-histidine, L-lysine, L-tryptophan, L-proline andL-arginine. L-homoserine likewise belongs to the L-amino acids.

The mutants of the invention preferably secrete said proteinogenic aminoacids, in particular L-lysine and L-tryptophan. The term amino acidsalso comprises their salts such as, for example, lysinemonohydrochloride or lysine sulfate in the case of the amino acidL-lysine.

The invention further relates to mutants of coryneform bacteria, whichcomprise a zwf allele encoding a polypeptide having glucose 6-phosphatedehydrogenase enzyme activity, which polypeptide comprises the aminoacid sequence of SEQ ID NO:2, with any proteinogenic amino acid otherthan glycine being present in position 321. Preference is given toreplacing glycine with L-serine. In addition, the amino acid sequence ofthe polypeptide comprises, where appropriate, replacement of the aminoacid L-serine in position 8 with a different proteinogenic amino acid,preferably L-threonine.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a zwf allele encoding a polypeptide having glucose6-phosphate dehydrogenase enzyme activity, which polypeptide comprisesany proteinogenic amino acid other than glycine, preferably L-serine, inthe position corresponding to position 321 of the amino acid sequence ofSEQ ID NO:2, the gene comprising a nucleotide sequence identical to thenucleotide sequence of a polynucleotide which is obtainable by apolymerase chain reaction (PCR) using a primer pair whose nucleotidesequences comprise in each case at least 15 contiguous nucleotidesselected from the nucleotide sequence between positions 1 and 307 of SEQID NO:3 or SEQ ID NO:11 and from the complementary nucleotide sequencebetween positions 2100 and 1850 of SEQ ID NO:3 or SEQ ID NO:11. Examplesof suitable primer pairs of this kind are depicted in SEQ ID NO:17 andSEQ ID NO:18 and in SEQ ID NO:19 and SEQ ID NO:20. The preferredstarting material (template DNA) is chromosomal DNA of coryneformbacteria which have been treated in particular with a mutagen.Particular preference is given to the chromosomal DNA of the genusCorynebacterium and very particular preference is given to that of thespecies Corynebacterium glutamicum.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a zwf allele encoding a polypeptide having glucose6-phosphate dehydrogenase enzyme activity, which polypeptide comprisesan amino acid sequence having a length corresponding to 514 L-aminoacids, with any proteinogenic amino acid other than glycine, preferablyL-serine, being present in position 321. In addition, the amino acidL-serine in position 8 of the amino acid sequence of the polypeptidehas, where appropriate, been replaced with a different proteinogenicamino acid, preferably L-threonine.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a zwf allele encoding a polypeptide having glucose6-phosphate dehydrogenase enzyme activity, which polypeptide comprisesthe amino acid sequence corresponding to positions 312 to 330 of SEQ IDNO:6 or 8 in positions 312 to 330 of the amino acid sequence.Preferably, the amino acid sequence of the encoded polypeptide comprisesan amino acid sequence corresponding to positions 307 to 335 of SEQ IDNO:6 or 8 or to positions 292 to 350 of SEQ ID NO:6 or 8 or to positions277 to 365 of SEQ ID NO:6 or 8 or to positions 262 to 380 of SEQ ID NO:6or 8 or to positions 247 to 395 of SEQ ID NO:6 or 8 or to positions 232to 410 of SEQ ID NO:6 or 8 or to positions 202 to 440 of SEQ ID NO:6 or8 or to positions 172 to 470 of SEQ ID NO:6 or 8 or to positions 82 to500 of SEQ ID NO:6 or 8 or to positions 2 to 512 of SEQ ID NO:6 or 8 orto positions 2 to 513 of SEQ ID NO:6 or 8 or to positions 2 to 514 ofSEQ ID NO:6 or 8. Very particular preference is given to the length ofthe encoded polypeptide comprising 514 amino acids.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a zwf allele encoding a polypeptide having glucose6-phosphate dehydrogenase enzyme activity, which polypeptide comprisesany amino acid other than glycine in position 321 or in thecorresponding position of the amino acid sequence, preference beinggiven to the substitution with L-serine, and whose amino acid sequenceis moreover at least 90%, preferably at least 92% or at least 94% or atleast 96%, and very particularly preferably at least 97% or at least 98%or at least 99%, identical to the amino acid sequence of SEQ ID NO:6. Anexample of an amino acid sequence having an identity of at least 99% tothe amino acid sequence of SEQ ID NO:6 is depicted in SEQ ID NO:8 and10. The polypeptide of this glucose 6-phosphate dehydrogenase possesses,apart from the amino acid substitution in position 321, the amino acidsubstitution of L-serine with L-threonine in position 8.

The invention furthermore relates to mutants of coryneform bacteria,which comprise a zwf allele encoding a polypeptide having glucose6-phosphate dehydrogenase enzyme activity, which polypeptide comprisesany amino acid other than glycine in position 321 or in thecorresponding position of the amino acid sequence, with preference beinggiven to the substitution with L-serine, and whose nucleotide sequenceis moreover at least 90%, preferably at least 92% or at least 94% or atleast 96%, and very particularly preferably at least 97% or at least 98%or at least 99%, identical to the nucleotide sequence of SEQ ID NO:5. Anexample of a nucleotide sequence of a zwf allele, which possesses atleast 99% identity to the nucleotide sequence of SEQ ID NO:5, isdepicted in SEQ ID NO:7. Apart from the nucleotide substitution ofguanine with allanine in position 961 (see SEQ ID NO:5), the nucleotidesequence of this zwf allele has the nucleotide substitution of thyminewith adenine in position 22 (see SEQ ID NO:7). A further example of anucleotide sequence of a zwf allele, which has at least 99% identity tothe nucleotide sequence of SEQ ID NO:5, is depicted in SEQ ID NO:9. Thenucleotide sequence of this zwf allele has, apart from the nucleotidesubstitution of guanine with adenine in position 961 (see SEQ ID NO:5)and the nucleotide substitution of thymine with adenine in position 22(see SEQ ID NO:7), the nucleotide substitutions of cytosine with thyminein position 138, of cytosine with thymine in position 279, of thyminewith cytosine in position 738, of cytosine with thymine in position 777and of guanine with adenine in position 906 (see SEQ ID NO:9).

Conservative amino acid substitutions are known to alter the enzymeactivity only insignificantly. Accordingly, the zwf allele which ispresent in the mutants of the invention and which encodes a polypeptidehaving glucose 6-phosphate dehydrogenase enzyme activity may compriseone (1) or more conservative amino acid substitution(s), in addition tothe amino acid sequence depicted in SEQ ID NO:6 and SEQ ID NO:8 and,respectively, SEQ ID NO:10. Preference is given to the polypeptidecomprising no more than two (2), no more than three (3), no more thanfour (4) or no more than five (5), conservative amino acidsubstitutions.

In the case of the aromatic amino acids, the substitutions are said tobe conservative when phenylalanine, tryptophan and tyrosine aresubstituted for one another. In the case of the hydrophobic amino acids,the substitutions are said to be conservative when leucine, isoleucineand valine are substituted for one another. In the case of the polaramino acids, the substitutions are said to be conservative whenglutamine and asparagine are substituted for one another.

In the case of the basic amino acids, the substitutions are said to beconservative when arginine, lysine and histidine are substituted for oneanother. In the case of the acidic amino acids, the substitutions aresaid to be conservative when aspartic acid and glutamic acid aresubstituted for one another. In the case of the hydroxylgroup-containing amino acids, the substitutions are said to beconservative when serine and threonine are substituted for one another.

An example of a conservative amino acid substitution is the substitutionof serine with threonine in position 8 of SEQ ID NO:6, which results inthe amino acid sequence according to SEQ ID NO:8 and SEQ ID NO:10,respectively.

During work on the present invention, comparison of the amino acidsequence using the Clustal program (Thompson et al., Nucleic AcidsResearch 22, 4637-4680 (1994)) revealed that the amino acid sequences ofglucose 6-phosphate dehydrogenase of various bacteria such as, forexample, Escherichia coli, Bacillus subtilis, Mycobacteriumtuberculosis, Mycobacterium bovis, Streptomyces coeliclor, Streptomycesavermitilis, Corynebacterium efficiens and Corynebacterium glutamicum,comprise a sequence motif consisting of the sequenceVal-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu, a sequence motif consisting of thesequence Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys, or a sequence motif consistingof the sequence Arg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg.The term “Ali” represents the amino acids Ala or Val, the term “Afa”represents the amino acids Lys or Thr, and the term “Bra” represents theamino acids Ile or Leu.

Accordingly, preference is given to those mutants of coryneformbacteria, which comprise a zwf allele encoding a polypeptide havingglucose 6-phosphate dehydrogenase enzyme activity, which polypeptidecomprises at least one amino acid sequence selected from the groupconsisting of Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu,Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys andArg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg and whichcomprises any amino acid other than lysine, preferably L-serine, inposition 321 or in the corresponding or comparable position of the aminoacid sequence. In addition, the amino acid sequence comprises, whereappropriate, an amino acid substitution of L-serine with a differentproteinogenic amino acid, preferably L-threonine, in position 8according to SEQ ID NO:2.

The amino acid sequence motif Val-Ile-Phe-Gly-Val-Thr-Gly-Asp-Leu ispresent, for example, in SEQ ID NO:6, 8 or 10 from positions 32 to 40.The amino acid sequence motif Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys ispresent, for example, in SEQ ID NO:6, 8 and, respectively, 10 fromposition 203 to 210. The amino acid sequence motifArg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Leu-Arg-Thr-Gly-Lys-Arg is present, forexample, in SEQ ID NO:6, 8 or 10 from positions 354 to 367.

Finally, the invention relates to mutants of coryneform bacteria, whichcomprise a zwf allele encoding a polypeptide having glucose 6-phosphatedehydrogenase enzyme activity, which polypeptide comprises the aminoacid sequence of SEQ ID NO:6 or SEQ ID NO:8 and, respectively, SEQ IDNO:10.

Enzymes intrinsic to the host, called aminopeptidases, are known toremove the terminal methionine during protein synthesis.

The term “a position corresponding to position 321 of the amino acidsequence” or “a position comparable to position 321 of the amino acidsequence” means the fact that insertion or deletion of a codon codingfor an amino acid in the N-terminal region (based on position 321 of SEQID NO:6, 8 or 10) of the encoded polypeptide formally increases, in thecase of an insertion, or decreases, in the case of a deletion, theindicated position and indicated length, in each case by one unit. Forexample, deletion of the AAC codon coding for the amino acidL-asparagine in position 4 of SEQ ID NO:6, 8 or 10 moves the L-serinefrom position 321 to position 320. The indicated length would then be:513 amino acids. In the same way, insertion or deletion of a codoncoding for an amino acid in the C-terminal region (based on position321) of the encoded polypeptide formally increases, in the case of aninsertion, or decreases, in the case of a deletion, the indicated lengthby one unit. Such comparable positions can readily be identified bycomparing the amino acid sequences in the form of an alignment, forexample with the aid of the Clustal program.

Insertions and deletions of this kind essentially do not affect theenzymic activity. “Essentially do not affect” means that the enzymicactivity of the variants mentioned differs from the activity of thepolypeptide having the amino acid sequence of SEQ ID NO:6 or 8 and,respectively, 10 by no more than 10%, no more than 7.5%, no more than5%, no more than 2.5% or no more than 1%.

Accordingly, the invention also relates to zwf alleles encodingpolypeptide variants of SEQ ID NO:6 or 8 and, respectively, 10, whichvariants comprise one or more insertion(s) or deletion(s). Thepolypeptide preferably comprises no more than 5, no more than 4, no morethan 3 or no more than 2 amino acid insertions or deletions.

The sequence motifs Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu, andArg-Ile-Asp-His-Tyr-Leu-Gly-Lys andArg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg are preferablynot disrupted by such insertions/deletions.

The mutants of the invention may be prepared by classical in-vivomutagenesis methods with cell populations of coryneform bacteria byusing mutagenic substances such as, for example,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), ethyl methanesulfonate(EMS), 5-bromouracil, or ultraviolet light. Mutagenesis methods aredescribed, for example, in Manual of Methods for General Bacteriology(Gerhard et al. (Eds.), American Society for Microbiology, Washington,D.C., USA, 1981) or in Tosaka et al. (Agricultural and BiologicalChemistry 42(4), 745-752 (1978)) or in Konicek et al. (FoliaMicrobiologica 33, 337-343 (1988)). Typical mutageneses using MNNGcomprise concentrations of from 50 to 500 mg/l or else higherconcentrations of up to a maximum of 1 g/l, an incubation time of from 1to 30 minutes at a pH of from 5.5 to 7.5. Under these conditions, thenumber of viable cells is reduced by a proportion of from approx. 50% to90% or approx. 50% to 99% or approx. 50% to 99.9% or more.

Mutants or cells are removed from the mutagenized cell population andpropagated. Preference is given to investigating, in a further step,their ability to secrete amino acids, preferably L-lysine orL-tryptophan, in a batch culture using a suitable nutrient medium.Suitable nutrient media and assay conditions are described, inter alia,in U.S. Pat. No. 6,221,636, in U.S. Pat. No. 5,840,551, in U.S. Pat. No.5,770,409, in U.S. Pat. No. 5,605,818, in U.S. Pat. No. 5,275,940 and inU.S. Pat. No. 4,224,409. Using suitable robots, as described, forexample, in Zimmermann et al. (VDI Berichte No. 1841, VDI-Verlag,Düsseldorf, Germany 2004, 439-443) or Zimmermann (Chemie IngenenieurTechnik 77 (4), 426-428 (2005)), it is possible to study a large numberof mutants in a short time. In this way, mutants are identified which,compared to the parent strain or non-mutagenized starting strain,secrete an increased amount of amino acids into the nutrient medium orthe cell interior. These include, for example, those mutants whose aminoacid secretion has increased by at least 0.5%.

Subsequently, DNA of the mutants is provided or isolated from the latterand the corresponding polynucleotide is synthesized with the aid of thepolymerase chain reaction using primer pairs which allow amplificationof the zwf gene or of the zwf allele of the invention or of the mutationof the invention in position 321. Preference is given to isolating theDNA from those mutants which secrete an increased amount of amino acids.

To this end, it is possible to select any primer pairs from thenucleotide sequence located upstream and downstream of the mutation ofthe invention and from the nucleotide sequence complementary thereto. Aprimer of a primer pair here preferably comprises at least 15, at least18, at least 20, at least 21 or at least 24, contiguous nucleotidesselected from the nucleotide sequence between positions 1 and 1267 ofSEQ ID NO:3 or SEQ ID NO:11. The corresponding second primer of a primerpair comprises at least 15, at least 18, at least 20, at least 21 or atleast 24, contiguous nucleotides selected from the complementarynucleotide sequence of positions 2100 and 1271 of SEQ ID NO:3 or SEQ IDNO:11. If it is desired to amplify the coding region, then the primerpair is preferably selected from the nucleotide sequence betweenpositions 1 and 307 of SEQ ID NO:3 or SEQ ID NO:11 and from thecomplementary nucleotide sequence between positions 2100 and 1850 of SEQID NO:3 or SEQ ID NO:11. If it is desired to amplify part of the codingregion, as indicated, for example, in SEQ ID NO:14 and 15, then theprimer pair is preferably selected from the nucleotide sequence betweenpositions 309 and 1267 of SEQ ID NO:3 or SEQ ID NO:11 and from thecomplementary nucleotide sequence between positions 1848 and 1271 of SEQID NO:3 or SEQ ID NO:11. Examples of suitable primer pairs are thezwf-K1 and zwf-K2 primer pair depicted under SEQ ID NO:17 and SEQ IDNO:18 or the zwf-L1 and zwf-L2 primer pair depicted under SEQ ID NO:19and SEQ ID NO:20. In addition, the primer may be provided withrecognition sites for restriction enzymes, with a biotin group orfurther accessories as described in the prior art. The total length ofthe primer is usually no more than 30, 40, 50 or 60 nucleotides.

Usually, thermostable DNA polymerases are employed in the preparation ofpolynucleotides by amplification of selected sequences such as the zwfallele of the invention from initially introduced DNA, for examplechromosomal DNA (template DNA), via amplification by means of PCR.Examples of DNA polymerases of this kind are Taq polymerase of Thermusaquaticus, which is sold, inter alia, by Qiagen (Hilden, Germany), Ventpolymerase of Thermococcus litoralis, sold, inter alia, by New EnglandBiolabs (Frankfurt, Germany), or Pfu polymerase of Pyrococcus furiosus,sold, inter alia, by Stratagene (La Jolla, USA). Preference is given topolymerases having proof-reading activity. Proof-reading activity meansthat these polymerases are capable of recognizing wrongly incorporatednucleotides and rectifying the error by renewed polymerization(Lottspeich and Zorbas, Bioanalytik, Spektrum Akademischer Verlag,Heidelberg, Germany (1998)). Examples of polymerases havingproof-reading activity are Vent polymerase and Pfu polymerase.

The conditions in the reaction mixture are set according to theinformation provided by the manufacturer. The polymerases are usuallysupplied by the manufacturer together with the customary buffer whichusually has concentrations of 10-100 mM Tris/HCl and 6-55 mM KCl at pH7.5-9.3. Magnesium chloride is added in a concentration of 0.5-10 mM, ifnot present in the buffer supplied by the manufacturer. Furthermore,deoxynucleoside triphosphates are added in a concentration of 0.1-16.6mM to the reaction mixture. The primers, in a final concentration of0.1-3 μM, and template DNA, in the optimal case from 10² to 10⁵ copies,are initially introduced into the reaction mixture. 10⁶ to 10⁷ copiesmay also be used. An amount of 2-5 units of the appropriate polymeraseis added to the reaction mixture. A typical reaction mixture has avolume of 20-100 μl.

Further additives which may be added to the reaction are bovine serumalbumin, Tween-20, gelatin, glycerol, formamide or DMSO (Dieffenbach andDveksler, PCR Primer—A Laboratory Manual, Cold Spring Harbor LaboratoryPress, USA 1995).

A typical PCR profile consists of three different, successively repeatedtemperature stages. Initially, the reaction is started by increasing thetemperature to 92° C.-98° C. for 4 to 10 minutes in order to denaturethe initially introduced DNA. This is followed repeatedly by first astep of denaturing the initially introduced DNA at approximately 92-98°C. for 10-60 seconds, then a step of 10-60 seconds of binding theprimers to the initially introduced DNA at a particular temperaturedependent on said primers (annealing temperature), which from experienceis from 50° C. to 60° C. and can be calculated for each primer pairindividually. Detailed information on this can be found by the skilledworker in Rychlik et al. (Nucleic Acids Research 18 (21): 6409-6412).Subsequently, a synthesis step of extending the initially introducedprimers (extension) at the activity optimum of the polymerase, indicatedin each case and usually in the range from 73° C. to 67° C., preferably72° C. to 68° C., depending on the polymerase. The duration of thisextension step depends on the performance of the polymerase and on thelength of the PCR product to be amplified. In a typical PCR, this steplasts 0.5-8 minutes, preferably 2-4 minutes. These three steps arerepeated 30 to 35 times, where appropriate up to 50 times. A final“extension” step of 4-10 minutes ends the reaction. The polynucleotidesprepared in this manner are also referred to as amplicons; the termnucleic acid fragment is likewise common.

Further instructions and information regarding PCR can be found by theskilled worker for example in the manual “PCR-Strategies” (Innis,Felfand and Sninsky, Academic Press, Inc., 1995), in the manual byDiefenbach and Dveksler “PCR Primer—a laboratory manual” (Cold SpringHarbor Laboratory Press, 1995), in the manual by Gait “Oligonucleotidesynthesis: A Practical Approach” (IRL Press, Oxford, UK, 1984) and inNewton and Graham “PCR” (Spektrum Akademischer Verlag, Heidelberg,Germany, 1994).

The nucleotide sequence is subsequently determined, for example by thechain termination method of Sanger et al. (Proceedings of the NationalAcademies of Sciences, U.S.A., 74, 5463-5467 (1977)) with themodifications indicated by Zimmermann et al. (Nucleic Acids Research 18,1067 pp (1990)), and the polypeptide encoded by said nucleotide sequenceis analyzed, in particular with respect to the amino acid sequence. Forthis purpose, the nucleotide sequence is entered into a program fortranslating DNA sequence into an amino acid sequence. Examples ofsuitable programs are the program “Patentin” which is available frompatent offices, for example the US Patent and Trademark Office (USPTO),or “Translate Tool” which is available on the ExPASy Proteomics Serveron the World Wide Web (Gasteiger et al., Nucleic Acids Research 31,3784-3788 (2003)).

In this way, mutants are identified whose zwf alleles encodepolypeptides having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptides comprise any proteinogenic amino acid other thanglycine in position 321 of the amino acid sequence or in thecorresponding or comparable position. Preference is given to thesubstitution with L-serine. In addition, the amino acid sequencecomprises, where appropriate, an amino acid substitution of L-serinewith a different proteinogenic amino acid, preferably L-threonine, inposition 8 or in the corresponding or comparable position.

Accordingly, the invention relates to a mutant of a coryneformbacterium, which is obtainable by the following steps:

-   -   a) treating a coryneform bacterium capable of secreting amino        acids with a mutagenic agent,    -   b) isolating and propagating the mutant generated in a),    -   c) preferably determining the ability of said mutant to secrete        in a medium or to accumulate in the cell interior at least 0.5%        more amino acid than the coryneform bacterium employed in a),    -   d) providing nucleic acid of the mutant obtained in b),    -   e) preparing a nucleic acid molecule/amplicon/nucleic acid        fragment, using the polymerase chain reaction, of the nucleic        acid from d) and of a primer pair consisting of a first primer        comprising at least 15 contiguous nucleotides selected from the        nucleotide sequence between positions 1 and 1267 of SEQ ID NO:3        or SEQ ID NO:11 and a second primer comprising at least 15        contiguous nucleotides selected from the complementary        nucleotide sequence between positions 2100 and 1271 of SEQ ID        NO:3 or 11,    -   f) determining the nucleotide sequence of the nucleic acid        molecule obtained in e) and determining the encoded amino acid        sequence,    -   g) comparing, where appropriate, the amino acid sequence        determined in f) with SEQ ID NO:6, 8 or 10, and    -   h) identifying a mutant comprising a polynucleotide which        encodes a polypeptide comprising any proteinogenic amino acid        other than glycine, preferably L-serine, in position 321 or a        comparable position, and which, where appropriate, comprises any        proteinogenic amino acid other than L-serine, preferably        L-threonine, in position 8 or a comparable position.

The mutants generated in this way typically comprise one (1) copy of thezwf allele described.

SEQ ID NO:5, 7 and 9 depict, by way of example, the coding regions ofzwf alleles of mutants of the invention. The coding region of the wildtype gene is depicted as SEQ ID NO:1. SEQ ID NO:1 comprises thenucleobase guanine in position 961, the nucleobase guanine in position962 and the nucleobase cytosine in position 963. SEQ ID NO:1 comprisesthe GGC codon, coding for the amino acid glycine, in positions 961 to963. SEQ ID NO:5 comprises the nucleobase adenine in position 961. Thisguanine-adenine transition results in the AGC codon, coding for theamino acid L-serine, in positions 961 to 963.

SEQ ID NO:1 comprises the nucleobase thymine in position 22, thenucleobase cytosine in position 23 and the nucleobase cytosine inposition 24. Accordingly, SEQ ID NO:1 comprises the TCC codon, codingfor the amino acid serine, in positions 22 to 24. SEQ ID NO:7 comprisesthe nucleobase adenine in position 22. This thymine-adenine transversionresults in the AGC codon, coding for the amino acid L-serine, inpositions 22 to 24.

In addition, the nucleotide sequences depicted in SEQ ID NO:5 and 7 maycomprise further base substitutions which have resulted from themutagenesis treatment but which do not manifest themselves in an alteredamino acid sequence. Such mutations are referred to in the art also assilent or neutral mutations. These silent mutations may likewise alreadybe present in the coryneform bacterium used for mutagenesis treatment.Examples of such silent mutations are the cytosine-thymine transition inposition 138, the cytosine-thymine transition in position 279, thethymine-cytosine transition in position 738, the cytosine-thyminetransition in position 777 and the guanine-adenine transition inposition 906, as depicted in SEQ ID NO:9.

The coryneform bacteria used for the mutagenesis preferably already havethe ability to secrete the desired amino acid into the surroundingnutrient medium or fermentation broth or to accumulate it in the cellinterior.

L-Lysine-producing coryneform bacteria typically possess afeedback-resistant or desensibilized aspartate kinase.Feedback-resistant aspartate kinases mean aspartate kinases which,compared to the wild type, have a lower sensitivity to the inhibition bymixtures of lysine and threonine or mixtures of AEC (aminoethylcysteine)and threonine or lysine alone or AEC alone. The genes or allelesencoding these desensibilized aspartate kinases are also referred to aslysC^(FBR) alleles. The prior art (Table 1) describes numerouslysC^(FBR) alleles encoding aspartate kinase variants which have aminoacid substitutions in comparison with the wild type protein. SEQ IDNO:21 depicts the coding region of the wild type lysC gene ofCorynebacterium glutamicum according to accession number AX756575 of theNCBI database, and SEQ ID NO:22 depicts the protein encoded by saidgene.

TABLE 1 lysC^(FBR) alleles encoding feedback-resistant aspartate kinasesName of Further Accession allele information Reference number lysC^(FBR)E05108 JP 1993184366-A E05108 (sequence 1) lysC^(FBR) E06825 lysC A279TJP 1994062866-A E06825 (sequence 1) lysC^(FBR) E06826 lysC A279T JP1994062866-A E06826 (sequence 2) lysC^(FBR) E06827 JP 1994062866-AE06827 (sequence 3) lysC^(FBR) E08177 JP 1994261766-A E08177(sequence 1) lysC^(FBR) E08178 lysC A279T JP 1994261766-A E08178(sequence 2) lysC^(FBR) E08179 lysC A279V JP 1994261766-A E08179(sequence 3) lysC^(FBR) E08180 lysC S301F JP 1994261766-A E08180(sequence 4) lysC^(FBR) E08181 lysC T308I JP 1994261766-A E08181(sequence 5) lysC^(FBR) E08182 JP 1994261766-A E08182 (sequence 6)lysC^(FBR) E12770 JP 1997070291-A E12770 (sequence 13) lysC^(FBR) E14514JP 1997322774-A E14514 (sequence 9) lysC^(FBR) E16352 JP 1998165180-AE16352 (sequence 3) lysC^(FBR) E16745 JP 1998215883-A E16745 (sequence3) lysC^(FBR) E16746 JP 1998215883-A E16746 (sequence 4) lysC^(FBR)I74588 US 5688671-A I74588 (sequence 1) lysC^(FBR) I74589 lysC A279T US5688671-A I74589 (sequence 2) lysC^(FBR) I74590 US 5688671-A I74590(sequence 7) lysC^(FBR) I74591 lysC A279T US 5688671-A I74591 (sequence8) lysC^(FBR) I74592 US 5688671-A I74592 (sequence 9) lysC^(FBR) I74593lysC A279T US 5688671-A I74593 (sequence 10) lysC^(FBR) I74594 US5688671-A I74594 (sequence 11) lysC^(FBR) I74595 lysC A279T US 5688671-AI74595 (sequence 12) lysC^(FBR) I74596 US 5688671-A I74596 (sequence 13)lysC^(FBR) I74597 lysC A279T US 5688671-A I74597 (sequence 14)lysC^(FBR) X57226 lysC S301Y EP0387527 X57226 Kalinowski et al.,Molecular and General Genetics 224: 317-324 (1990) lysC^(FBR) L16848lysC G345D Follettie and L16848 Sinskey NCBI Nucleotide Database (1990)lysC^(FBR) L27125 lysC R320G Jetten et al., L27125 lysC G345D AppliedMicrobiology Biotechnology 43: 76-82 (1995) lysC^(FBR) lysC T311IWO0063388 (sequence 17) lysC^(FBR) lysC S301F US3732144 lysC^(FBR) lysCS381F EP0435132 lysC^(FBR) lysC S317A US5688671 (sequence 1) lysC^(FBR)lysC T380I WO 01/49854

L-Lysine-secreting coryneforme bacteria typically possess one or more ofthe amino acid substitutions listed in Table 1.

Preference is given to the following lysC^(FBR) alleles: lysC A279T(substitution of alanine in position 279 of the encoded aspartate kinaseprotein according to SEQ ID NO:22 with threonine), lysC A279V(substitution of alanine in position 279 of the encoded aspartate kinaseprotein according to SEQ ID NO:22 with valine), lysC S301F (substitutionof serine in position 301 of the encoded aspartate kinase proteinaccording to SEQ ID NO:22 with phenylalanine), lysC T308I (substitutionof threonine in position 308 of the encoded aspartate kinase proteinaccording to SEQ ID NO:22 with isoleucine), lysC S301Y (substitution ofserine in position 308 of the encoded aspartate kinase protein accordingto SEQ ID NO:22 with tyrosine), lysC G345D (substitution of glycine inposition 345 of the encoded aspartate kinase protein according to SEQ IDNO:22 with asparaginic acid), lysC R320G (substitution of arginine inposition 320 of the encoded aspartate kinase protein according to SEQ IDNO:22 with glycine), lysC T311I (substitution of threonine in position311 of the encoded aspartate kinase protein according to SEQ ID NO:22with isoleucine), lysC S381F (substitution of serine in position 381 ofthe encoded aspartate kinase protein according to SEQ ID NO:22 withphenylalanine) and lysC S317A (substitution of serine in position 317 ofthe encoded aspartate kinase protein according to SEQ ID NO:22 withalanine).

Particular preference is given to the lysC^(FBR) allele lysC T311I(substitution of threonine in position 311 of the encoded aspartatekinase protein according to SEQ ID NO:22 with isoleucine) and alysC^(FBR) allele comprising at least one substitution selected from thegroup consisting of A279T (substitution of alanine in position 279 ofthe encoded aspartate kinase proteins according to SEQ ID NO:22 withthreonine) and S317A (substitution of serine in position 317 of theencoded aspartate kinase protein according to SEQ ID NO:22 withalanine).

The lysC^(FBR) allele lysC T311I is present in the strain DM1797deposited with the DSMZ. DM1797 is a mutant of Corynebacteriumglutamicum ATCC13032.

Starting from strain DM1797, a mutant referred to as DM1816, whichharbors a zwf allele encoding a polypeptide in which L-serine is presentin position 321 of the amino acid sequence, was isolated in the mannerdescribed above. The nucleotide sequence of the coding region of the zwfallele of the DM1816 mutant is depicted as SEQ ID NO:9 and the aminoacid sequence of the encoded polypeptide is depicted as SEQ ID NO:10 and12, respectively. The DM1816 mutant additionally comprises nucleotidesubstitutions in the nucleotide sequence between positions 1 and 307 ofSEQ ID NO:3. These nucleotide substitutions are depicted in SEQ IDNO:11. SEQ ID NO:11 comprises guanine instead of adenine in position208, adenine instead of guanine in position 235, cytosine instead ofthymine in position 245, guanine instead of adenine in position 257 andguanine instead of adenine in position 299. SEQ ID NO:11 furthermorecomprises adenine instead of thymine in position 329, thymine instead ofcytosine in position 445, thymine instead of cytosine in position 586,cytosine instead of thymine in position 1045, thymine instead ofcytosine in position 1084, adenine instead of guanine in position 1213and adenine instead of guanine in position 1268.

In addition it is possible to use L-lysine-secreting coryneform bacteriawhich have an attenuated homoserine dehydrogenase or homoserine kinaseor which possess other properties as known from the prior art.

L-Tryptophan-producing coryneform bacteria typically possess afeedback-resistant or desensibilized anthranilate synthase. The termfeedback-resistant anthranilate synthase means anthranilate synthaseswhich, compared to the wild type, have a lower sensitivity to inhibition(5% to 10%, 10% to 15% or 10% to 20%) by tryptophan or5-fluorotryptophan (Matsui et al., Journal of Bacteriology 169 (11):5330-5332 (1987)) or similar analogs. The genes or alleles encodingthese desensibilized anthranilate synthases are also referred to astrpE^(FBR) alleles. Examples of mutants or alleles of this kind aredescribed, for example, in U.S. Pat. No. 6,180,373 and EP0338474.

The mutants obtained show increased secretion or production of thedesired amino acid in a fermentation process, in comparison with thestarting strain or parent strain employed.

The invention likewise relates to an isolated polynucleotide encoding apolypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises any proteinogenic amino acid other thanglycine in position 321 or in a corresponding or comparable position ofthe amino acid sequence, with preference being given to the substitutionwith L-serine.

The polynucleotide of the invention may be isolated from a mutant of theinvention.

It is furthermore possible to use in-vitro methods for the mutagenesisof the zwf gene. The use of in-vitro methods involves subjectingisolated polynucleotides which comprise a zwf gene of a coryneformbacterium, preferably the Corynebacterium glutamicum wild type genedescribed in the prior art, to a mutagenic treatment.

The isolated polynucleotides may be, for example, isolated total DNA orchromosomal DNA or else amplicons of the zwf gene, which have beenprepared with the aid of the polymerase chain reaction (PCR). Suchamplicons are also referred to as PCR products. Instructions for theamplification of DNA sequences with the aid of the polymerase chainreaction can be found by the skilled worker, inter alia, in the manualby Gait: Oligonucleotide Synthesis: A Practical Approach (IRL Press,Oxford, UK, 1984) and Newton and Graham: PCR (Spektrum AkademischerVerlag, Heidelberg, Germany, 1994). It is likewise possible toincorporate the zwf gene to be mutagenized first into a vector, forexample into a bacteriophage or into a plasmid.

Suitable methods of in-vitro mutagenesis are, inter alia, the treatmentwith hydroxylamine according to Miller (Miller, J. H.: A Short Course inBacterial Genetics. A Laboratory Manual and Handbook for Escherichiacoli and Related Bacteria, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1992), the use of mutagenic oligonucleotides (T. A.Brown: Gentechnologie fur Einsteiger [Genetic engineering forbeginners], Spektrum Akademischer Verlag, Heidelberg, 1993 and R. M.Horton: PCR-Mediated Recombination and Mutagenesis, MolecularBiotechnology 3, 93-99 (1995)) and the use of a polymerase chainreaction using a DNA polymerase with a high error rate. An example ofsuch a DNA polymerase is the Mutazyme DNA Polymerase (GeneMorph PCRMutagenesis Kit, No. 600550) from Stratagene (La Jolla, Calif., USA).

Further instructions and reviews on the generation of mutations in vivoor in vitro can be found in the prior art and in known textbooks ofgenetics and molecular biology, such as, for example, the textbook byKnippers (“Molekulare Genetik”, 6th edition, Georg Thieme Verlag,Stuttgart, Germany, 1995), that by Winnacker (“Gene and Klone”, VCHVerlagsgesellschaft, Weinheim, Germany, 1990) or that by Hagemann(“Allgemeine Genetik”, Gustav Fischer Verlag, Stuttgart, 1986).

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises the amino acid sequence of SEQ ID NO:2, withany proteinogenic amino acid other than glycine being present inposition 321 of said amino acid sequence. Preference is given to thesubstitution with L-serine. In addition, the amino acid sequence of thepolypeptide comprises, where appropriate, an amino acid substitution ofL-serine with a different amino acid, preferably L-threonine, inposition 8.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises an amino acid sequence having a length of514 amino acids, with any proteinogenic L-amino acid other than glycine,preferably L-serine, being present in position 321.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises, from position 312 to 330 of the amino acidsequence, the amino acid sequence corresponding to positions 312 to 330of SEQ ID NO:6 or 8. The amino acid sequence of the encoded polypeptidepreferably comprises an amino acid sequence corresponding to positions307 to 335 of SEQ ID NO:6 or 8 or to positions 292 to 350 of SEQ ID NO:6or 8 or to positions 277 to 365 of SEQ ID NO:6 or 8 or to positions 262to 380 of SEQ ID NO:6 or 8 or to positions 247 to 395 of SEQ ID NO:6 or8 or to positions 232 to 410 of SEQ ID NO:6 or 8 or to positions 202 to440 of SEQ ID NO:6 or 8 or to positions 172 to 470 of SEQ ID NO:6 or 8or to positions 82 to 500 of SEQ ID NO:6 or 8 or to positions 2 to 512of SEQ ID NO:6 or 8 or to positions 2 to 513 of SEQ ID NO:6 or 8 or topositions 2 to 514 of SEQ ID NO:6 or 8. The length of the encodedpolypeptide comprises very particularly preferably 514 amino acids.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises any proteinogenic amino acid other thanglycine, preferably L-serine, in position 321 of the amino acid sequenceor in a corresponding or comparable position, and comprising anucleotide sequence identical to the nucleotide sequence of apolynucleotide which is obtainable by a polymerase chain reaction (PCR)using the primer pair whose nucleotide sequences comprise in each caseat least 15 contiguous nucleotides selected from the nucleotide sequencebetween positions 1 and 307 of SEQ ID NO:3 or SEQ ID NO:11 and from thecomplementary nucleotide sequence between positions 2100 and 1850 of SEQID NO:3 or SEQ ID NO:11. Examples of suitable primer pairs of this kindare depicted in SEQ ID NO:17 and SEQ ID NO:18 and in SEQ ID NO:19 andSEQ ID NO:20. The preferred starting material (template DNA) ischromosomal DNA of coryneform bacteria, in particular of those whichhave been treated with a mutagen. Particular preference is given to thechromosomal DNA of the genus Corynebacterium, and very particularpreference is given to that of the species Corynebacterium glutamicum.

The invention furthermore relates to an isolated polynucleotide whichhybridizes with the nucleotide sequence complementary to SEQ ID NO:5, 7or 9 under stringent conditions and which encodes a polypeptide havingglucose 6-phosphate dehydrogenase enzyme activity, which polypeptidecomprises any proteinogenic amino acid other than glycine, preferablyL-serine, in position 321 of the amino acid sequence or in acorresponding or comparable position and, where appropriate, anyproteinogenic amino acid other than L-serine, preferably L-threonine, ina position corresponding to position 8.

Instructions regarding the hybridization of nucleic acids orpolynucleotides can be found by the skilled worker, inter alia, in themanual “The DIG System User's Guide for Filter Hybridization” fromBoehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl et al.(International Journal of Systematic Bacteriology 41: 255-260 (1991)).The hybridization is carried out under stringent conditions, i.e. onlyhybrids in which the probe, i.e. a polynucleotide comprising thenucleotide sequence complementary to SEQ ID NO:5, 7 or 9, and the targetsequence, i.e. the polynucleotides treated or identified with the probe,are at least 90% identical, are formed. The stringency of thehybridization, including that of the washing steps, is known to beinfluenced or determined by varying the buffer composition, thetemperature and the salt concentration. The hybridization reaction isgenerally carried out at relatively low stringency compared to thewashing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington,UK, 1996).

For example, a buffer corresponding to 5×SSC buffer at a temperature ofapprox. 50° C.-68° C. may be used for the hybridization reaction. Inthis case, probes may also hybridize with polynucleotides which are lessthan 90% identical to the nucleotide sequence of the probe employed.Such hybrids are less stable and are removed by washing under stringentconditions. This may be achieved, for example, by lowering the saltconcentration to 2×SSC and, where appropriate, subsequently to 0.5×SSC(The DIG System User's Guide for Filter Hybridisation, BoehringerMannheim, Mannheim, Germany, 1995), with the temperature being set toapprox. 50° C.-68° C., approx. 52° C.-68° C., approx. 54° C.-68° C.,approx. 56° C.-68° C., approx. 58° C.-68° C., approx. 60° C.-68° C.,approx. 62° C.-68° C., approx. 64° C.-68° C., approx. 66° C.-68° C.Temperature ranges of approx. 64° C.-68° C. or approx. 66° C.-68° C. arepreferred. It is possible, where appropriate, to lower the saltconcentration down to a concentration corresponding to 0.2×SSC or0.1×SSC. The SSC buffer comprises, where appropriate, sodium dodecylsulfate (SDS) in a concentration of 0.1%. By gradually increasing thehybridization temperature in steps of approx. 1-2° C. from 50° C. to 68°C., it is possible to isolate polynucleotide fragments which have atleast 90% or at least 91%, preferably at least 92% or at least 93% or atleast 94% or at least 95% or at least 96%, and very particularlypreferably at least 97% or at least 98% or at least 99%, identity to thesequence or complementary sequence of the probe employed and whichencode a polypeptide which has glucose 6-phosphate dehydrogenase enzymeactivity and comprises the amino acid substitution of the invention. Thenucleotide sequence of the polynucleotide obtained in this way isdetermined by known methods. Further instructions regardinghybridization are commercially available in the form of “kits” (e.g. DIGEasy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, Catalog No.1603558). The nucleotide sequences thus obtained encode polypeptideshaving glucose 6-phosphate dehydrogenase enzyme activity, whichpolypeptides are at least 90%, preferably at least 92% or at least 94%or at least 96%, and very particularly preferably at least 97% or atleast 98% or at least 99%, identical to the amino acid sequence of SEQID NO:6 or SEQ ID NO:8 and which comprise the amino acid substitution ofthe invention.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises any amino acid other than glycine inposition 321 or in a corresponding or comparable position of the aminoacid sequence, the substitution with L-serine being preferred, and whichcomprises an amino acid sequence which moreover is at least 90%,preferably at least 92% or at least 94% or at least 96%, and veryparticularly preferably at least 97% or at least 98% or at least 99%,identical to the amino acid sequence of SEQ ID NO:6. One example of apolypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises an amino acid sequence at least 99%identical to that of SEQ ID NO:6, is depicted in SEQ ID NO:8 and SEQ IDNO:10.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises any amino acid other than glycine inposition 321 or in a corresponding or comparable position of the aminoacid sequence, the substitution with L-serine being preferred, andcomprising a nucleotide sequence which moreover is at least 90%,preferably at least 92% or at least 94% or at least 96%, and veryparticularly preferably at least 97% or at least 98% or at least 99%,identical to the nucleotide sequence of SEQ ID NO:5. An example of apolynucleotide which encodes a polypeptide of the invention havingglucose 6-phosphate dehydrogenase enzyme activity and which has anucleotide sequence at least 99% identical to that of SEQ ID NO:5 isdepicted in SEQ ID NO:7. The nucleotide sequence of this zwf allele has,in addition to the nucleotide substitution of guanine with adenine inposition 961 (see SEQ ID NO:5), the nucleotide substitution of thyminewith adenine in position 22 (see SEQ ID NO:7). Another example of anucleotide sequence of a zwf allele, which has at least 99, identity tothe nucleotide sequence of SEQ ID NO:5, is depicted in SEQ ID NO:9. Thenucleotide sequence of this zwf allele has, in addition to thenucleotide substitution of guanine with adenine in position 961 (see SEQID NO:5) and the nucleotide substitution of thymine with adenine inposition 22 (see SEQ ID NO:7), the nucleotide substitutions of cytosinewith thymine in position 138, of cytosine with thymine in position 279,of thymine with cytosine in position 738, of cytosine with thymine inposition 777, and of guanine with adenine in position 906 (see SEQ IDNO:9).

In addition, preference is given to those isolated polynucleotidesencoding a polypeptide having glucose 6-phosphate dehydrogenase enzymeactivity, which polypeptide comprises any amino acid other than glycine,preferably L-serine, in position 321 of the amino acid sequence or in acorresponding or comparable position, and comprising at least onesequence motif or an amino acid sequence selected from the groupconsisting of Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leu,Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys, andArg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg.

The term “Ali” represents the amino acids Ala or Val, the term “Afa”represents the amino acids Lys or Thr, and the term “Bra” represents theamino acids Ile or Leu.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises the amino acid sequence of SEQ ID NO:6 or 8and, respectively, 10. The encoded polypeptide comprises, whereappropriate, one (1) or more conservative amino acid substitution(s).Preferably, the polypeptide comprises no more than two (2), no more thanthree (3), no more than four (4) or no more than five (5), conservativeamino acid substitutions.

The invention furthermore relates to an isolated polynucleotide encodinga polypeptide having glucose 6-phosphate dehydrogenase enzyme activity,which polypeptide comprises the amino acid sequence of SEQ ID NO:6 or 8and, respectively, 10, including an extension at the N- or C-terminus byat least one (1) amino acid. This extension has no more than 50, 40, 30,20, 10, 5, 3 or 2 amino acids or amino acid residues.

Finally, the invention also relates to zwf alleles encoding polypeptidevariants of SEQ ID NO:6, 8 or 10, which comprise one or more insertionsor deletions. These preferably comprise no more than 5, no more than 4,no more than 3 or no more than 2 insertions or deletions of amino acids.Preferably, the sequence motifs Val-Ile-Phe-Gly-Ali-Afa-Gly-Asp-Leuand/or Arg-Ile-Asp-His-Tyr-Leu-Gly-Lys and/orArg-Trp-Ala-Gly-Val-Pro-Phe-Tyr-Bra-Arg-Thr-Gly-Lys-Arg are notdisrupted by such insertions/deletions.

The invention furthermore relates to an isolated polynucleotidecomprising the nucleotide sequence according to SEQ ID NO:5, 7, 9 or 11.

The invention furthermore relates to an isolated polynucleotidecomprising the nucleotide sequence between positions 1 and 307 of SEQ IDNO:11, preferably the nucleotide sequence between positions 198 and 304of SEQ ID NO:11, and very particularly preferably the nucleotidesequence between positions 208 and 299 of SEQ ID NO:11.

Finally, the invention relates to an isolated polynucleotide comprisingthe zwf allele of the DM1816 mutant.

Moreover, the invention relates to an isolated polynucleotide comprisingpart of the coding region of a zwf allele of the invention, saidisolated polynucleotide comprising in any case that part of the codingregion which comprises the amino acid substitution in position 321 ofthe amino acid sequence of the encoded polypeptide.

More specifically, a nucleic acid molecule or DNA fragment is comprisedwhich encodes at least one amino acid sequence corresponding topositions 307 to 335 of SEQ ID NO:2 or which encodes at least one aminoacid sequence corresponding to positions 292 to 350 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 277 to 365 of SEQ ID NO:2 or which encodes at least one aminoacid sequence corresponding to positions 262 to 380 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 247 to 395 of SEQ ID NO:2 or which encodes at least one aminoacid sequence corresponding to positions 232 to 410 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 202 to 440 of SEQ ID NO:2, or which encodes at least one aminoacid sequence corresponding to positions 172 to 470 of SEQ ID NO:2 orwhich encodes at least one amino acid sequence corresponding topositions 82 to 500 of SEQ ID NO:2, or which encodes at least one aminoacid sequence corresponding to positions 2 to 512 of SEQ ID NO:2, orwhich encodes at least one amino acid sequence corresponding topositions 2 to 513 of SEQ ID NO:2, or which encodes at least one aminoacid sequence corresponding to positions 2 to 514 of SEQ ID NO:2, orwhich includes a corresponding reading frame, with any proteinogenicamino acid other than glycine, preferably L-serine, being present in theposition corresponding to 321 of SEQ ID NO:2 and, where appropriate, anyproteinogenic amino acid except L-serine, preferably L-threonine, beingpresent in the position corresponding to 8.

An example of a reading frame of the invention, comprising apolynucleotide encoding at least the amino acid sequence of positions307 to 335 corresponding to SEQ ID NO:2, with any proteinogenic aminoacid (Xaa) other than glycine being present in the positioncorresponding to 321 of the amino acid sequence, is listed below:

gat aaa acc tcc gct cgt ggt cag tac gct gcc ggt Asp Lys Thr Ser Ala ArgGly Gln Tyr Ala Ala Gly 307         310                 315 tgg cag TrpGln     320 nnn tct gag tta gtc aag gga ctt cgc gaa gaa gat Xaa Ser GluLeu Val Lys Gly Leu Arg Glu Glu Asp                325                 330 ggc ttc aac Gly Phe Asn        335

It is likewise depicted as SEQ ID NO:13. The amino acid sequence encodedby this reading frame is depicted as SEQ ID NO:14. Position 15 in SEQ IDNO:14 corresponds to position 321 of SEQ ID NO:6, 8, 10 or 12.

Preference is given to nucleic acid molecules encoding at least oneamino acid sequence corresponding to positions 307 to 335 of SEQ ID NO:6or 8 and, respectively, 10, or at least corresponding to position 292 to350 of SEQ ID NO:6 or 8 and, respectively, 10, or at least correspondingto positions 277 to 365 of SEQ ID NO:6 or 8 and, respectively, 10, or atleast corresponding to positions 262 to 380 of SEQ ID NO:6 or 8 and,respectively, 10, or at least corresponding to positions 247 to 395 ofSEQ ID NO:6 or 8 and, respectively, 10, or at least corresponding topositions 232 to 410 of SEQ ID NO:6 or 8 and, respectively, 10, or atleast corresponding to positions 202 to 440 of SEQ ID NO:6 or 8 and,respectively, 10, or at least corresponding to positions 172 to 470 ofSEQ ID NO:6 or 8 and, respectively, 10, or at least corresponding topositions 82 to 500 of SEQ ID NO:6 or 8 and, respectively, 10, or atleast corresponding to positions 2 to 512 of SEQ ID NO:6 or 8 and,respectively, 10, or at least corresponding to positions 2 to 513 of SEQID NO:6 or 8 and, respectively 10, or at least corresponding topositions 2 to 514 of SEQ ID NO:6 or 8 and, respectively, 10.

An example of a reading frame of the invention, comprising apolynucleotide encoding at least the amino acid sequence correspondingto positions 307 to 335 of SEQ ID NO:6, is listed below:

gat aaa acc tcc gct cgt ggt cag tac gct gcc ggt Asp Lys Thr Ser Ala ArgGly Gln Tyr Ala Ala Gly 307         310                 315 tgg cag TrpGln     320 agc tct gag tta gtc aag gga ctt cgc gaa gaa gat Ser Ser GluLeu Val Lys Gly Leu Arg Glu Glu Asp                325                 330 ggc ttc aac Gly Phe Asn        335

The reading frame is likewise depicted as SEQ ID NO:15. SEQ ID NO:16depicts the amino acid sequence encoded by said reading frame. Position15 in SEQ ID NO:16 corresponds to position 321 of SEQ ID NO:6, 8, 10 or12.

Very particular preference is given to nucleic acid molecules comprisingat least one nucleotide sequence corresponding to positions 919 to 1005of SEQ ID NO:5, 7 or 9, or at least one nucleotide sequencecorresponding to positions 874 to 1050 of SEQ ID NO:5, 7 or 9, or atleast one nucleotide sequence corresponding to positions 829 to 1095 ofSEQ ID NO:5, 7 or 9, or at least one nucleotide sequence correspondingto positions 784 to 1140 of SEQ ID NO:5, 7 or 9, or at least onenucleotide sequence corresponding to positions 739 to 1185 of SEQ IDNO:5, 7 or 9, or at least one nucleotide sequence corresponding topositions 694 to 1230 of SEQ ID NO:5, 7 or 9, or at least one nucleotidesequence corresponding to positions 604 to 1320 of SEQ ID NO:5, 7 or 9,or at least one nucleotide sequence corresponding to positions 514 to1410 of SEQ ID NO:5, 7 or 9, or at least one nucleotide sequencecorresponding to positions 244 to 1500 of SEQ ID NO:5, 7 or 9, or atleast one nucleotide sequence corresponding to positions 4 to 1536 ofSEQ ID NO:5, 7 or 9, or at least one nucleotide sequence correspondingto positions 4 to 1539 of SEQ ID NC:5, 7 or 9, or at least onenucleotide sequence corresponding to positions 4 to 1542 of SEQ ID NO:5,7 or 9.

In addition, the reading frames of the invention, as shown by way ofexample in SEQ ID NO:13 and 15 as nucleotide sequence and in SEQ IDNO:14 and SEQ ID NO:16 in the form of the encoded amino acid sequence,may comprise one or more mutations resulting in one or more conservativeamino acid substitutions. The mutations preferably result in no morethan 4%, no more than 2% or no more than 1%, conservative amino acidsubstitutions. The reading frames of the invention may furthermorecomprise one more silent mutations. The reading frames of the inventioncomprise preferably no more than 4%, and particularly preferably no morethan 2% to no more than 1%, silent mutations.

The isolated polynucleotides of the invention may be used in order toproduce recombinant strains of microorganisms, which release amino acidsinto the surrounding medium or accumulate them in the cell interior inan improved manner, compared to the starting or parent strain.

A widespread method of incorporating mutations into genes of coryneformbacteria is that of allele substitution which is also referred to asgene replacement. This process involves transferring a DNA fragmentcomprising the mutation of interest into the desired strain of acoryneform bacterium and incorporating said mutation into the chromosomeof the desired strain by at least two recombination events or cross-overevents or replacing the sequence of a gene in the strain in questionwith the mutated sequence.

Schwarzer and Pühler (Bio/Technology 9, 84-87 (1991) used this method inorder to incorporate a lysA allele carrying a deletion and a lysA allelecarrying an insertion into the C. glutamicum chromosome, instead of thewild type gene. Schäfer et al. (Gene 145, 69-73 (1994)) employed saidmethod in order to incorporate a deletion into the C. glutamicumhom-thrB operon. Nakagawa et al. (EP 1108790) and Ohnishi et al.(Applied Microbiology and Biotechnology 58(2), 217-223 (2002)) employedsaid method in order to incorporate various mutations, starting from theisolated alleles, into the C. glutamicum chromosome. In this way,Nakagawa et al. succeeded in incorporating a mutation referred to asVal59Ala into the homoserine dehydrogenase gene (hom), a mutationreferred to as Thr311Ile into the aspartate kinase gene (lysC and ask,respectively), a mutation referred to as Pro458Ser into the pyruvatecarboxylase gene (pyc) and a mutation referred to as Ala213Thr into theglucose 6-phoshate dehydrogenase gene (zwf) of C. glutamicum strains.

A process of the invention may use a polynucleotide of the invention,which comprises the entire coding region, as depicted, for example, inSEQ ID NO:5, 7 or 9, or which comprises part of the coding region, suchas, for example, the nucleotide sequence encoding at least the aminoacid sequence corresponding to positions 307 to 335 of SEQ ID NO:6, 8 or10, and depicted as SEQ ID NO:13 and 15. The part of the coding regioncorresponding to SEQ ID NO:13 and 15 is ≧87 nucleobases in length.Preference is given to those parts of the coding region whose length is≧267 nucleobases, such as, for example, nucleic acid molecules encodingat least one amino acid sequence corresponding to positions 277 to 365of SEQ ID NO:6, 8 or 10. Very particular preference is given to thoseparts of the coding region whose length is ≧357 nucleobases, such as,for example, nucleic acid molecules coding for at least one amino acidsequence corresponding to positions 262 to 380 of SEQ ID NO:6, 8 or 10.

In said method, the DNA fragment comprising the mutation of interest istypically present in a vector, in particular a plasmid which preferablyis replicated only to a limited extent, if at all, by the strain to beprovided with the mutation. The auxiliary or intermediate host used, inwhich the vector can be replicated, is usually a bacterium of the genusEscherichia, preferably of the species Escherichia coli.

Examples of plasmid vectors of this kind are the pK*mob and pK*mobsacBvectors described by Schäfer et al. (Gene 145, 69-73 (1994)), such as,for example, pK18mobsacB, and the vectors described in WO 02/070685 andWO 03/014362. These are replicative in Escherichia coli but not incoryneform bacteria. Particularly suitable are vectors comprising a genewith a conditionally negative-dominant action, such as, for example, thesacB gene (levansucrase gene) of Bacillus, for example, or the galK gene(galactose kinase gene) of Escherichia coli, for example. (A gene withconditionally negative-dominant action means a gene which, under certainconditions, is disadvantageous, for example toxic, to the host but whichhas, under different conditions, no adverse effects on the host carryingthe gene.) Said vectors make possible the selection for recombinationevents in which the vector is eliminated from the chromosome. Nakamuraet al. (U.S. Pat. No. 6,303,383) furthermore described atemperature-sensitive plasmid for coryneform bacteria, which canreplicate only at temperatures below 31° C.

The vector is subsequently transferred to the coryneform bacterium byway of conjugation, for example by the method of Schäfer (Journal ofBacteriology 172, 1663-1666 (1990)), or transformation, for example bythe method of Dunican and Shivnan (Bio/Technology 7, 1067-1070 (1989))or the method of Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)). The DNA may also be transferred,where appropriate, by particle bombardment.

Incorporation of the mutation is achieved after homologous recombinationby means of a first cross-over event causing integration and of asuitable second cross-over event causing excision in the target gene orin the target sequence, resulting in a recombinant bacterium.

The strains obtained may be identified and characterized by using, interalia, the methods of Southern blotting hybridization, polymerase chainreaction, sequence determination, the method of fluorescence resonanceenergy transfer (FRET) (Lay et al. Clinical Chemistry 43, 2262-2267(1997)) or methods of enzymology.

Accordingly, the invention further relates to a process for preparing acoryneform bacterium, which comprises

-   -   a) transferring a polynucleotide of the invention to a        coryneform bacterium,    -   b) replacing the glucose 6-phosphate dehydrogenase gene which        encodes an amino acid sequence with glycine in position 321 or        in a comparable position of said amino acid sequence and which        is present in the chromosome of said coryneform bacterium with        the polynucleotide of a), which encodes an amino acid sequence        having a different L-amino acid, preferably L-serine, in        position 321 or in a comparable position of said amino acid        sequence and, where appropriate, any proteinogenic amino acid        other than L-serine, preferably the amino acid L-threonine, in        position 8 or in a comparable position, and    -   c) propagating the coryneform bacterium obtained by steps a) and        b).

In this way a recombinant coryneform bacterium is obtained whichcomprises one (1) zwf allele of the invention, instead of the wild typezwf gene.

Another process of the invention for preparing a microorganism comprises

-   -   a) transferring a polynucleotide of the invention, which encodes        a polypeptide having glucose 6-phosphate dehydrogenase enzyme        activity, to a microorganism,    -   b) replicating said polynucleotide in said microorganism, and    -   c) propagating the microorganism obtained by steps a) and b).

In this way a recombinant microorganism is obtained, which comprises atleast one (1) copy or several copies of a polynucleotide of theinvention, which polynucleotide encodes a glucose 6-phosphatedehydrogenase comprising any proteinogenic amino acid other than glycinein position 321 or a comparable position of the amino acid sequence ofthe encoded polypeptide, the substitution with L-serine being preferred.The polypeptide comprises, where appropriate, any proteinogenic aminoacid other than L-serine, preferably the amino acid L-threonine, inposition 8 or a comparable position.

Accordingly, the invention further relates to hosts or host cells,preferably microorganisms, particularly preferably coryneform bacteriaand bacteria of the genus Escherichia, which comprise thepolynucleotides of the invention. The invention likewise relates tomicroorganisms prepared by using the isolated polynucleotides. Suchmicroorganisms or bacteria are also referred to as recombinantmicroorganisms or recombinant bacteria. In the same way, the inventionrelates to vectors comprising the polynucleotides of the invention.Finally, the invention likewise relates to hosts harboring said vectors.

The isolated polynucleotides of the invention may likewise be used forachieving overexpression of the polypeptides encoded by them.

Overexpression generally means an increase in the intracellularconcentration or activity of a ribonucleic acid, a protein or an enzyme.In the case of the present invention, zwf alleles or polynucleotideswhich encode glucose 6-phosphate dehydrogenases comprising anyproteinogenic amino acid other than glycine in position 321 of the aminoacid sequence of the encoded polypeptide, with the substitution withL-serine being preferred, are overexpressed. The encoded proteinmoreover comprises, where appropriate, a substitution of L-serine with adifferent proteinogenic amino acid, preferably L-threonine, in position8 of the amino acid sequence. Enzymes endogenous to thehost—“aminopeptidases”—are known to be able to cleave N-terminal aminoacids, in particular the N-terminal methionine, off the polypeptideproduced. Said increase in the concentration or activity of a geneproduct can be achieved, for example, by increasing the copy number ofthe corresponding polynucleotides by at least one copy.

A widespread method of increasing the copy number comprisesincorporating the appropriate gene or allele into a vector, preferably aplasmid, which is replicated by a coryneform bacterium. Examples ofsuitable plasmid vectors are pZ1 (Menkel et al., Applied andEnvironmental Microbiology (1989) 64: 549-554) or the pSELF vectorsdescribed by Tauch et al. (Journal of Biotechnology 99, 79-91 (2002)). Areview article on plasmids in Corynebacterium glutamicum can be found inTauch et al. (Journal of Biotechnology 104, 27-40 (2003)).

Another common method of achieving overexpression is the process ofchromosomal gene amplification. This method involves inserting at leastone additional copy of the gene or allele of interest into thechromosome of a coryneform bacterium.

In one embodiment, as described, for example, for the hom-thrB operon inReinscheid et al. (Applied and Environmental Microbiology 60, 126-132(1994)), a plasmid which is non-replicative in C. glutamicum and whichcomprises the gene of interest is transferred to a coryneform bacterium.After homologous recombination by means of a cross-over event, theresulting strain comprises at least two copies of the gene or allele inquestion.

In another embodiment described in WO 03/040373 and US-2003-0219881-A1,one or more copies of the gene of interest are inserted at a desiredside of the C. glutamicum chromosome by means of at least tworecombination events. In this way, for example, a copy of a lysC alleleencoding a L-lysine-insensitive aspartate kinase was incorporated intothe C. glutamicum gluB gene.

In a further embodiment described in WO 03/014330 andUS-2004-0043458-A1, at least one further copy, preferably in tandemarrangement to the gene or allele already present, of the gene ofinterest is incorporated by means of at least two recombinantion eventsat the natural locus. In this way it was possible, for example, toachieve a tandem duplication of a lysC^(FBR) allele at the natural lysCgene locus.

Another method of achieving overexpression comprises linking theappropriate gene or allele functionally (operably linked) to a promoteror an expression cassette.

Examples of suitable promoters for Corynebacterium glutamicum aredescribed in the review article by Patek et al. (Journal ofBiotechnology 104(1-3), 311-323 (2003)). It is furthermore possible touse the well-known promoters T3, T7, SP6, M13, lac, tac and trcdescribed by Amann et al. (Gene 69(2), 301-315 (1988)) and Amann andBrosius (Gene 40(2-3), 183-190 (1985)). Such a promotor may be inserted,for example, upstream of the zwf allele, typically at a distance ofapproximately 1-500 or 1-307 nucleotides from the start codon, of arecombinant coryneform bacterium, which allele comprises, instead of theamino acid glycine naturally present in position 321, a differentproteinogenic amino acid. A promotor of this kind may naturally likewisebe inserted upstream of the zwf allele of a mutant of the invention. Itis furthermore possible to link an isolated polynucleotide of theinvention, which encodes a variant of the invention of glucose6-phosphate dehydrogenase, to a promotor and to incorporate theexpression unit obtained into an extrachromosomally replicating plasmidor into the chromosome of a coryneform bacterium.

In addition, it is possible to mutate the promotor and regulatoryregions or the ribosomal binding site which is located upstream of thestructural gene. An example of a mutated promotor region of the zwf geneor zwf allele is the nucleotide sequence comprising positions 208 to 299of SEQ ID NO:11. Measures of extending the mRNA lifetime likewiseimprove expression. Preventing the degradation of the enzyme proteinfurthermore likewise enhances enzyme activity. Alternatively, the geneor allele in question may furthermore be overexpressed by altering themedia composition and the culturing process.

The overexpression measures increase the activity or concentration ofthe protein in question usually by at least 10%, 25%, 50%, 75%, 100%,150%, 200%, 300%, 400% or 500%, up to no more than 1000% or 2000%, basedon the activity or concentration of the protein in the startingmicroorganism or parent strain. A starting microorganism or parentstrain means a microorganism which is subjected to the measures of theinvention.

A method of determining the enzymic activity of glucose 6-phosphatedehydrogenase is described in Moritz et al. (European Journal ofBiochemistry 267, 3442-3452 (2000)).

The concentration of the protein may be determined via 1- and2-dimensional protein gel fractionation and subsequent opticalidentification of the protein concentration in the gel, usingappropriate evaluation software. A common method of preparing theprotein gels in the case of coryneform bacteria and of identifying theproteins is the procedure described by Hermann et al. (Electrophoresis,22:1712-23 (2001)). The protein concentration may likewise be determinedby Western blot hybridization using an antibody specific for the proteinto be detected (Sambrook et al., Molecular cloning: a laboratory manual.2^(nd) Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989) and subsequent optical evaluation using appropriateconcentration determination software (Lohaus and Meyer (1998)Biospektrum 5:32-39; Lottspeich, Angewandte Chemie 321: 2630-2647(1999)).

Accordingly, the invention relates to processes for overexpressing theglucose 6-phosphate dehydrogenases of the invention. A process of theinvention for overexpression comprises, inter alia, increasing the copynumber of a polynucleotide of the invention, which polynucleotideencodes a glucose 6-phosphate-dehydrogenase variant comprising anyproteinogenic amino acid other than glycine in position 321 or thecorresponding position of the encoded amino acid sequence andcomprising, where appropriate, any proteinogenic amino acid other thanL-serine in position 8 or the corresponding position, by at least one(1) or several copies. Another process of the invention comprisesfunctionally linking a promotor to the polynucleotide.

The invention furthermore relates to microorganisms having an increasedconcentration or activity of the glucose 6-phosphate dehydrogenasevariants of the invention in their cell interior.

It may be additionally advantageous for improved production of L-aminoacids to overexpress in the mutants or recombinant strains of theinvention one or more enzymes of the particular biosynthetic pathway, ofglycolysis, of anaplerotics, of the citrate cycle, of the pentosephosphate cycle, of the amino acid export and, where appropriate,regulatory proteins. Preference is usually given to the use ofendogenous genes.

“Endogenous genes” or “endogenous nucleotide sequences” means the genesor the nucleotide sequences or alleles present in the population of aspecies.

Thus it is possible to overexpress for the preparation of L-lysine oneor more of the genes selected from the group consisting of

-   -   a dapA gene encoding a dihydrodipicolinate synthase, such as,        for example, the Corynebacterium glutamicum wild-type dapA gene        described in EP 0 197 335,    -   a zwf gene encoding a glucose 6-phosphate dehydrogenase, such        as, for example, the Corynebacterium glutamicum wild-type zwf        gene described in JP-A-09224661 and EP-A-1108790,    -   the Corynebacterium glutamicum zwf alleles described in        US-2003-0175911-A1, which encode a protein in which, for        example, the L-alanine in position 243 of the amino acid        sequence has been replaced with L-threonine or in which the        L-aspartic acid in position 245 has been replaced with L-serine,    -   a pyc gene encoding a pyruvate carboxylase, such as, for        example, the Corynebacterium glutamicum wild-type pyc gene        described in DE-A-198 31 609 and EP 1108790,    -   the Corynebacterium glutamicum pyc allele described in EP 1 108        790, which encodes a protein in which L-proline in position 458        of the amino acid sequence has been replaced by L-serine,    -   the Corynebacterium glutamicum pyc alleles described in WO        02/31158, which encode proteins which, according to claim 1,        carry one or more of the amino acid substitutions selected from        the group consisting of L-glutamic acid in position 153 replaced        with L-aspartic acid, L-alanine in position 182 replaced with        L-serine, L-alanine in position 206 replaced with L-serine,        L-histidine in position 227 replaced with L-arginine, L-alanine        in position 452 replaced with glycine and L-aspartic acid in        position 1120 replaced with L-glutamic acid (FIG. 2A in WO        02/31158 specifies two different start positions for the        pyruvate carboxylase, which positions differ by a length        corresponding to 17 amino acids. Accordingly, position 153 in        accordance with claim 1 in WO 02/31158 corresponds to a position        170 in FIG. 2A in WO 02/31158, while position 182 in accordance        with claim 1 corresponds to position 199 in FIG. 2A, position        206 in accordance with claim 1 corresponds to position 223 in        FIG. 2A, position 227 in accordance with claim 1 corresponds to        position 244 in FIG. 2A, position 452 in accordance with claim 1        corresponds to position 469 in FIG. 2A, position 1120 in        accordance with claim 1 corresponds to position 1137 in FIG. 2B.        FIG. 2A in WO 02/31158 furthermore indicates an amino acid        substitution of A (alanine) with G (glycine) in position 472.        Position 472 of the protein having the N terminal sequence MTA        corresponds to position 455 of the protein having the N-terminal        sequence MST according to FIG. 2A. FIG. 2B in WO 02/31158        furthermore indicates an amino acid substitution of D (aspartic        acid) with E (glutamic acid) in position 1133 of the protein        having the N-terminus MTA.),    -   a lysC gene encoding an aspartate kinase, such as, for example,        that of Corynebacterium glutamicum wild-type lysC gene described        as SEQ ID NO:281 in EP-A-1108790 (see also accession numbers        AX120085 and 120365) and that of Corynebacterium glutamicum        wild-type lysC gene, described as SEQ ID NO:25 in WO 01/00843        (see accession number AX063743),    -   a lysC^(FBR) allele, in particular according to Table 1, which        encodes a feedback-resistant aspartate kinase variant,    -   a lysE gene encoding a lysine export protein, such as, for        example, the Corynebacterium glutamicum wild-type lysE gene        described in DE-A-195 48 222,    -   the Corynebacterium glutamicum wild-type zwa1 gene encoding the        Zwa1 protein (U.S. Pat. No. 6,632,644).

In addition to using the alleles of the invention of the zwf gene, itmay also be advantageous, for the purpose of producing L-lysine, tosimultaneously attenuate or eliminate one or more of the endogenousgenes selected from the group consisting of

-   -   a pgi gene encoding glucose 6-phosphate isomerase, such as, for        example, the Corynebacterium glutamicum pgi gene described in        U.S. Pat. No. 6,586,214 and U.S. Pat. No. 6,465,238,    -   a hom gene encoding homoserine dehydrogenase, such as, for        example, the Corynebacterium glutamicum hom gene described in        EP-A-0131171,    -   a thrB gene encoding homoserine kinase, such as, for example,        the Corynebacterium glutamicum thrB gene described by Peoples et        al. (Molecular Microbiology 2 (1988): 63-72)), and    -   a pfkB gene encoding phosphofructokinase, such as, for example,        the Corynebacterium glutamicum pfkB gene described in WO        01/00844 (sequence no. 57).

In this connection, the term “attenuation” describes the reduction orelimination of the intracellular activity of one or more enzymes(proteins) which are encoded by the corresponding DNA in a microorganismwhich is achieved, for example, by using a weak promoter or using a geneor allele which encodes a corresponding enzyme having low activity, orinactivating the corresponding gene or enzyme (protein), and, whereappropriate, combining these measures.

As a result of using the measures for achieving attenuation, theactivity or concentration of the corresponding protein is generallylowered to from 0 to 75%, from 0 to 50%, from 0 to 25%, from 0 to 10%,or from 0 to 5%, of the activity or concentration of the wild-typeprotein or of the activity or concentration of the protein in thestarting microorganism.

Mutations which come into consideration for generating an attenuationare transitions, transversions, insertions and deletions of at least one(1) base pair or nucleotide. Depending on the effect which the aminoacid substitution elicited by the mutation has on the enzyme activity,reference is made to missense mutations or nonsense mutations. Amissense mutation leads to the replacement of a given amino acid in aprotein with another amino acid, with the amino acid replacementconstituting, in particular, a nonconservative amino acid substitution.This substitution impairs the efficiency or activity of the protein andreduces it down to a value of from 0 to 75%, from 0 to 50%, from 0 to25%, from 0 to 10%, or from 0 to 5%. A nonsense mutation leads to a stopcodon being located in the coding region of the gene and consequently totranslation being terminated prematurely. Insertions or deletions of atleast one base pair in a gene lead to frame shift mutations which resultin incorrect amino acids being incorporated or in the translation beingterminated prematurely. If a stop codon is formed in the coding regionas a consequence of mutation, this then also leads to translation beingterminated prematurely.

Directions for generating such mutations belong to the prior art and arecontained in known textbooks of genetics and molecular biology such asthe textbook by Knippers (“Molekulare Genetik [Molecular Genetics]”,6^(th) edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), that byWinnacker (“Gene und Klone [Genes and Clones]”, VCH Verlagsgesellschaft,Weinheim, Germany, 1990) or that by Hagemann (“Allgemeine Genetik[General Genetics]”, Gustav Fischer Verlag, Stuttgart, 1986). Furthermeasures are described in the prior art.

The isolated coryneform bacteria which are obtained by the measures ofthe invention exhibit a secretion or production of the desired aminoacid, in a fermentation process, which is increased as compared withthat of the starting strain or parent strain which was initiallyemployed.

“Isolated bacteria” are to be understood as being the mutants andrecombinant bacteria, in particular coryneform bacteria, according tothe invention which are isolated or generated and which comprise a zwfallele which encodes a glucose 6-phosphate dehydrogenase which comprisesthe described amino acid substitution in position 321 of the amino acidsequence and, where appropriate, an amino acid substitution of L-serinewith another proteinogenic amino acid, preferably L-threonine, inposition 8.

The performance of the isolated bacteria, or of the fermentation processwhen using these bacteria, in regard to one or more of the parametersselected from the group comprising the product concentration (productper volume), the product yield (product formed per carbon sourceconsumed) and the product formation (product formed per volume andtime), or else of other process parameters and combinations, is improvedby at least 0.5%, at least 1%, at least 1.5%, or at least 2%, based onthe starting strain or parent strain or the fermentation process whenusing these strains.

The isolated coryneform bacteria according to the invention can becultured continuously, as described, for example, in PCT/EP2004/008882,or discontinuously, in a batch process or a fed-batch process or arepeated fed-batch process, for the purpose of producing L-amino acids.A general summary of known culturing methods can be found in thetextbook by Chmiel (Bioprozesstechnik 1. Einfuhrung in dieBioverfahrenstechnik [Bioprocess Technology 1. Introduction toBioprocess Technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or inthe textbook by Storhas (Bioreaktoren und periphere Einrichtungen[Bioreactors and Peripheral Equipment] (Vieweg Verlag,Brunswick/Wiesbaden, 1994)).

The culture medium or fermentation medium to be used must suitablysatisfy the requirements of the given strains. Descriptions of media forculturing different microorganisms are given in the manual “Manual ofMethods for General Bacteriology” published by the American Society forBacteriology (Washington D.C., USA, 1981). The terms culture medium andfermentation medium or medium are mutually interchangeable.

The carbon source employed can be sugars and carbohydrates, such asglucose, sucrose, lactose, fructose, maltose, molasses,sucrose-containing solutions derived from sugar beet or sugar caneproduction, starch, starch hydrolysate and cellulose, oils and fats,such as soybean oil, sunflower oil, peanut oil and coconut oil, fattyacids, such as palmitic acid, stearic acid and linoleic acid, alcohols,such as glycerol, methanol and ethanol, and organic acids, such asacetic acid. These substances can be used individually or as mixtures.

The nitrogen source employed can be organic nitrogen-containingcompounds, such as peptones, yeast extract, meat extract, malt extract,cornsteep liquor, soybean flour and urea, or inorganic compounds, suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources can be usedindividually or as mixtures.

The phosphorus source employed can be phosphoric acid, potassiumdihydrogen phosphate or dipotassium hydrogen phosphate or thecorresponding sodium-containing salts.

The culture medium must furthermore contain salts, for example in theform of chlorides or sulfates of metals such as sodium, potassium,magnesium, calcium and iron, for example magnesium sulfate or ironsulfate, which are necessary for growth. Finally, essential growthsubstances, such as amino acids, for example homoserine, and vitamins,for example thiamine, biotin or pantothenic acid, can be used inaddition to the abovementioned substances. In addition to this, suitableprecursors of the respective amino acid can be added to the culturemedium.

The abovementioned added substances can be added to the culture in theform of a once-only mixture or fed in in a suitable manner during theculture.

Basic compounds such as sodium hydroxide, potassium hydroxide, ammoniaor ammonia water, or acidic compounds such as phosphoric acid orsulfuric acid, are employed in a suitable manner for controlling the pHof the culture. In general, the pH is adjusted to a value of from 6.0 to9.0, preferably of from 6.5 to 8. It is possible to use antifoamants,such as fatty acid polyglycol esters, for controlling foam formation.Suitable substances which act selectively, such as antibiotics, can beadded to the medium in order to maintain the stability of plasmids. Inorder to maintain aerobic conditions, oxygen or oxygen-containing gasmixtures, such as air, are passed into the culture. It is also possibleto use liquids which are enriched with hydrogen peroxide. Whereappropriate, the fermentation is conducted under positive pressure, forexample under a pressure of 0.03 to 0.2 MPa. The temperature of theculture is normally from 20° C. to 45° C., and preferably from 25° C. to40° C. In the case of batch processes, the culture is continued until amaximum of the desired amino acid has been formed. This objective isnormally achieved within from 10 hours to 160 hours. Longer culturingtimes are possible in the case of continuous processes.

Suitable fermentation media are described, inter alia, in U.S. Pat. No.6,221,636, U.S. Pat. No. 5,840,551, U.S. Pat. No. 5,770,409, U.S. Pat.No. 5,605,818, U.S. Pat. No. 5,275,940 and U.S. Pat. No. 4,224,409.

Methods for determining L-amino acids are disclosed in the prior art.The analysis can, for example, take place by means of anion exchangechromatography, followed by ninhydrin derivatization, as described inSpackman et al. (Analytical Chemistry, 30 (1958), 1190), or it can takeplace by reversed phase HPLC, as described in Lindroth et al.(Analytical Chemistry (1979) 51: 1167-1174).

The invention accordingly relates to a process for preparing an L-aminoacid, which comprises

-   -   a) fermenting an isolated coryneform bacterium in a suitable        medium, said bacterium comprising a gene encoding a polypeptide        having glucose 6-phosphate dehydrogenase enzyme activity, with        the glycine in position 321 or the corresponding position in the        amino acid sequences of said polypeptide having been replaced by        a different proteinogenic amino acid, preferably L-serine, and        with, where appropriate, the L-serine in position 8 or the        corresponding position having been replaced with a different        proteinogenic amino acid, preferably L-threonine, and    -   b) the L-amino acid being accumulated in the fermentation broth        or in the cells of the isolated coryneform bacterium.

The fermentation broth which has been prepared in this way is thensubjected to further processing into a solid or liquid product.

A fermentation broth is understood as being a fermentation medium inwhich a microorganism is cultured for a certain time and at a certaintemperature. The fermentation medium, and/or the medium employed duringthe fermentation, contains/contain all the substances or componentswhich ensure propagation of the microorganism and the formation of thedesired amino acid.

At the conclusion of the fermentation, the resulting fermentation brothaccordingly contains a) the biomass of the microorganism which has beenformed as a consequence of the propagation of the cells of themicroorganism, b) the desired amino acid which has been formed duringthe fermentation, c) the organic by-products which have been formedduring the fermentation, and d) the constituents of the fermentationmedium/fermentation media employed, or the added substances, for examplevitamins, such as biotin, amino acids, such as homoserine, or salts,such as magnesium sulfate, which were not consumed by the fermentation.

The organic by-products include substances which are produced by themicroorganisms employed in the fermentation, where appropriate inaddition to the given desired L-amino acid, and are secreted, whereappropriate. These by-products include L-amino acids which amount toless than 30%, 20% or 10% compared with the desired amino acid. Theyalso include organic acids which carry from one to three carboxylgroups, such as acetic acid, lactic acid, citric acid, malic acid orfumaric acid. Finally, they also include sugars, such as trehalose.

Typical fermentation broths which are suitable for industrial purposeshave an amino acid content of from 40 g/kg to 180 g/kg or of from 50g/kg to 150 g/kg. In general, the content of biomass (as dry biomass) isfrom 20 to 50 g/kg.

In the case of the amino acid L-lysine, essentially four differentproduct forms have been disclosed in the prior art.

One group of L-lysine-containing products comprises concentrated,aqueous, alkaline solutions of purified L-lysine (EP-B-0534865). Anothergroup, as described, for example, in U.S. Pat. No. 6,340,486 and U.S.Pat. No. 6,465,025, comprises aqueous, acidic, biomass-containingconcentrates of L-lysine-containing fermentation broths. The best-knowngroup of solid products comprises pulverulent or crystalline forms ofpurified or pure L-lysine, which is typically present in the form of asalt such as L-lysine monohydrochloride. Another group of solid productforms is described, for example, in EP-B-0533039. The product form whichis described in this document contains, in addition to L-lysine, themajor portion of the added substances which were used during thefermentative preparation, and which were not consumed, and, whereappropriate, from >0% to 100% of the biomass of the microorganismemployed.

In correspondence with the different product forms, a very wide varietyof methods are known for collecting, isolating or purifying the L-aminoacid from the fermentation broth for the purpose of preparing theL-amino acid-containing product or the purified L-amino acid.

It is essentially ion exchange chromatography methods, where appropriateusing active charcoal, and crystallization methods which are used forpreparing solid, pure L-amino acids. In the case of lysine, this resultsin the corresponding base or a corresponding salt such as themonohydrochloride (Lys-HCl) or the lysine sulfate (Lys₂-H₂SO₄).

As far as lysine is concerned, EP-B-0534865 describes a method forpreparing aqueous, basic L-lysine-containing solutions from fermentationbroths. In this document, the biomass is separated off from thefermentation broth and discarded. A base such as sodium hydroxide,potassium hydroxide or ammonium hydroxide is used to adjust the pH tobetween 9 and 11. Following concentration and cooling, the mineralconstituents (inorganic salts) are separated off from the broth bycrystallization and either used as fertilizer or discarded.

In the case of processes for preparing lysine using the bacteriaaccording to the invention, preference is given to those processes whichresult in products which contain constituents of the fermentation broth.These products are, in particular, used as animal feed additives.

Depending on the requirement, the biomass can be entirely or partiallyremoved from the fermentation broth by means of separation methods suchas centrifugation, filtration or decanting, or a combination of thesemethods, or all the biomass can be left in the fermentation broth. Whereappropriate, the biomass, or the biomass-containing fermentation broth,is inactivated during a suitable process step, for example by means ofthermal treatment (heating) or by means of adding acid.

In one approach, the biomass is completely or virtually completelyremoved, such that no (0%) or at most 30%, at most 20%, at most 10%, atmost 5%, at most 1% or at most 0.1%, of the biomass remains in theprepared product. In another approach, the biomass is not removed, oronly removed in trivial amounts, such that all (100%) or more than 70%,80%, 90%, 95%, 99% or 99.9% of the biomass remains in the preparedproduct. In one process according to the invention, the biomass isaccordingly removed in proportions of from ≧0% to ≦100%.

Finally, the fermentation broth which is obtained after the fermentationcan be adjusted, before or after the biomass has been completely orpartially removed, to an acid pH using an inorganic acid, such ashydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid,such as propionic acid (GB 1,439,728 or EP 1 331 220). It is likewisepossible to acidify the fermentation broth when it contains the entirebiomass (U.S. Pat. No. 6,340,486 or U.S. Pat. No. 6,465,025). Finally,the broth can also be stabilized by adding sodium bisulfite (NaHSO₃, GB1,439,728) or another salt, for example an ammonium, alkali metal oralkaline earth metal salt of sulfurous acid.

Organic or inorganic solids which may be present in the fermentationbroth are partially or entirely removed when the biomass is separatedoff. At least some (>0%), preferably at least 25%, particularlypreferably at least 50%, and very particularly preferably at least 75%,of the organic by-products which are dissolved in the fermentation brothand the constituents of the fermentation medium (added substances),which are dissolved and not consumed remain in the product. Whereappropriate, these by-products and constituents also remain completely(100%) or virtually completely, that is >95% or >98%, in the product. Inthis sense, the term “fermentation broth basis” means that a productcomprises at least a part of the constituents of the fermentation broth.

Subsequently, water is extracted from the broth, or the broth isthickened or concentrated, using known methods, for example using arotary evaporator, a thin-film evaporator or a falling-film evaporator,or by means of reverse osmosis or nanofiltration. This concentratedfermentation broth can then be worked up into flowable products, inparticular into a finely divided powder or, preferably, a coarse-grainedgranulate, using methods of freeze drying, of spray drying or of spraygranulation, or using other methods, for example in a circulatingfluidized bed as described in PCT/EP2004/006655. Where appropriate, adesired product is isolated from the resulting granulate by means ofscreening or dust separation.

It is likewise possible to dry the fermentation broth directly, i.e. byspray drying or spray granulation without any prior concentration.

“Flowable” is understood as meaning powders which discharge unhinderedfrom a series of glass discharge vessels having discharge apertures ofdifferent sizes, i.e. which discharge unhindered at least from thevessel having a 5 mm (millimeter) aperture (Klein: Seifen, Öle, Fette,Wachse [Soaps, Oils, Fats and Waxes] 94, 12 (1968)).

“Finely divided” means a powder the majority (>50%) of which has aparticle size which is from 20 to 200 μm in diameter.

“Coarse-grained” means a product the majority (>50%) of which has aparticle size of from 200 to 2000 μm in diameter.

The particle size can be determined using methods of laser diffractionspectrometry. The corresponding methods are described in the textbook“Teilchengröβenmessung in der Laborpraxis [Particle Size Measurement inLaboratory Practice]” by R. H. Müller and R. Schuhmann,Wissenschaftliche Verlagsgesellschaft Stuttgart (1996) or in thetextbook “Introduction to Particle Technology” by M. Rhodes, Wiley &Sons (1998).

The flowable, finely divided powder can in turn be converted, by meansof suitable compacting or granulating methods, into a coarse-grained,readily flowable, storable, and to a large extent dust-free, product.

The term “dust-free” means that the product only contains smallproportions (<5%) of particle sizes of less than 100 μm in diameter.

Within the meaning of this invention, “storable” means a product whichcan be stored for at least one (1) year or longer, preferably at least1.5 years or longer, particularly preferably two (2) years or longer, ina dry and cool environment without there being any significant loss(<5%) of the given amino acid.

The invention accordingly also relates to a process for preparing anL-amino acid-, preferably L-lysine- or L-tryptophan-, containingproduct, preferably an animal feed additive, from fermentation broths,which process is characterized by the steps of

-   -   a) culturing and fermenting an L-amino acid-secreting coryneform        bacterium, which comprises at least one zwf allele encoding a        polypeptide having glucose 6-phosphate dehydrogenase activity,        which polypeptide comprises an amino acid sequence in which any        proteinogenic amino acid other than glycine, preferably        L-serine, is present in position 321 or the comparable position,        with, where appropriate, any proteinogenic amino acid other than        L-serine, preferably L-threonine, being present in position 8 or        the comparable position, in a fermentation medium,    -   b) removing from 0 to 100% by weight of the biomass which is        formed during the fermentation, and    -   c) drying the fermentation broth which is obtained in accordance        with a) and/or b) in order to obtain the product in the desired        powder form or granulate form,        with, where appropriate, an acid selected from the group        sulfuric acid, phosphoric acid or hydrochloric acid being added        prior to step b) or c).

Preference is given to water being removed (concentration) from theL-amino acid-containing fermentation broth after step a) or b).

It is advantageous to use customary organic or inorganic auxiliarysubstances, or carrier substances such as starch, gelatin, cellulosederivatives or similar substances, as are customarily used as binders,gelatinizers or thickeners in foodstuff or feedstuff processing, orother substances, such as silicic acids, silicates (EP0743016A) orstearates, in connection with the granulation or compacting.

It is furthermore advantageous to provide the surface of the resultinggranulates with oils, as described in WO 04/054381. The oils which canbe used are mineral oils, vegetable oils or mixtures of vegetable oils.Examples of these oils are soybean oil, olive oil and soybeanoil/lecithin mixtures. In the same way, silicone oils, polyethyleneglycols or hydroxyethyl cellulose are also suitable. Treating thesurfaces with said oils increases the abrasion resistance of the productand reduces the dust content. The content of oil in the product is from0.02 to 2.0% by weight, preferably from 0.02 to 1.0% by weight, and veryparticularly preferably from 0.2 to 1.0% by weight, based on the totalquantity of the feedstuff additives.

Preference is given to products having a content of ≧97% by weight of aparticle size of from 100 to 1800 μm, or a content of ≧95% by weight ofa particle size of from 300 to 1800 μm, in diameter. The content ofdust, i.e. particles having a particle size of <100 μm, is preferablyfrom >0 to 1% by weight, particularly preferably at most 0.5% by weight.

Alternatively, however, the product can also be absorbed onto an organicor inorganic carrier substance which is known and customary in feedstuffprocessing, for example silicic acids, silicates, grists, brans, meals,starches, sugars etc., and/or be mixed and stabilized with customarythickeners or binders. Application examples and methods in this regardare described in the literature (Die Mühle+Mischfuttertechnik [TheGrinding Mill+Mixed Feed Technology] 132 (1995) 49, page 817).

Finally, the product can also be brought, by means of coating methodsusing film formers such as metal carbonates, silicic acids, silicates,alginates, stearates, starches, rubbers and cellulose ethers, asdescribed in DE-C-4100920, into a state in which it is stable towardsdigestion by animal stomachs, in particular the ruminant stomach.

In order to set a desired amino acid concentration in the product, theappropriate amino acid can, depending on the requirement, be addedduring the process in the form of a concentrate or, where appropriate,of a largely pure substance or its salt in liquid or solid form. Thelatter can be added individually, or as mixtures, to the resultingfermentation broth, or to the concentrated fermentation broth, or elsebe added during the drying process or granulation process.

In the case of lysine, the ratio of the ions is adjusted during thepreparation of lysine-containing products such that the ion ratio inaccordance with the following formula2×[SO₄ ²⁻]+[Cl⁻]−[NH₄ ⁺]−[Na⁺]−[K⁺]−2×[Mg⁺]−2×[Ca²⁺]/[L-Lys]has a value of from 0.68 to 0.95, preferably of from 0.68 to 0.90, asdescribed by Kushiki et al. in US 20030152633.

In the case of lysine, the solid fermentation broth-based product whichhas been prepared in this way has a lysine content (as lysine base) offrom 10% by weight to 70% by weight or of from 20% by weight to 70% byweight, preferably of from 30% by weight to 70% by weight and veryparticularly preferably of from 40% by weight to 70% by weight, based onthe dry mass of the product. It is also possible to achieve maximumcontents of lysine base of 71% by weight, 72% by weight or 73% byweight.

In the case of an electrically neutral amino acid such as L-tryptophan,the solid fermentation broth-based product which has been prepared inthis way has an amino acid content of at least 5% by weight, 10% byweight, 20% by weight or 30% by weight and maximally 50% by weight, 60%by weight, 70% by weight, 80% by weight, 90% by weight or up to 95% byweight.

The water content of the solid product is up to 5% by weight, preferablyup to 4% by weight, and particularly preferably less than 3% by weight.

A mutant of Corynebacterium glutamicum which is designated DM1797 andwhich comprises the amino acid substitution lysC T311I in its aspartatekinase was deposited on Oct. 28, 2004 with the Deutsche Samnlung furMikroorganismen und Zellkulturen [German Collection of Microorganismsand Cell Cultures] (DSMZ, Brunswick, Germany) as DSM 16833.

The Corynebacterium glutamicum mutant DM1816 of the invention, whichcomprises L-serine in position 321 in the amino acid sequence of the Zwfpolypeptide, was deposited on Feb. 9, 2005 at the Deutsche Sammlung furMikroorganismen und Zellkulturen [German Collection of Microorganismsand Cell Cultures] (DSMZ, Brunswick, Germany) as DSM17119.

EXAMPLE 1

Mutagenesis of the L-Lysine-Producing Strain DM1797

The Corynebacterium glutamicum strain DM1797 was used as a startingstrain for mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG).The DM1797 strain is an aminoethylcysteine-resistant mutant ofCorynebacterium glutamicum ATCC13032 and has been deposited under thename DSM16833 with the Deutsche Sammlung für Mikroorganismen undZellkulturen (DSMZ, Brunswick, Germany).

The DM1797 strain was cultured in 10 ml of LB broth (Merck, Darmstadt,Germany) contained in a 100 ml Erlenmeyer flask on a Certomat BS-1rotary shaker (B. Braun Biotech International, Melsungen, Germany) at33° C. and 200 rpm for 24 hours. The culture was subsequently removed bycentrifugation, the sediment was resuspended in 10 ml of 0.9% NaClsolution, the suspension obtained was again removed by centrifugationand the sediment obtained was taken up in 10 ml of 0.9% NaCl solution. 5ml of this cell suspension were treated with 400 μg/ml MNNG on a shaker(see above) at 30° C. and 200 rpm for 15 minutes. The mutagenesismixture was subsequently removed by centrifugation and the sediment wastaken up in 10 ml of 2% sodium thiosulfate in 0.9% NaCl buffer (pH=6.0).The cell suspension was then diluted 1:1000, 1:10000 and 1:100000 with0.9% NaCl solution, and aliquots were plated on brain-heart agar (Merck,Darmstadt, Germany). Approximately 2500 mutants were isolated in thisway.

EXAMPLE 2

Performance Test of the DM1797 Strain Mutants

The mutants obtained in example 1 were cultured in a nutrient mediumsuitable for lysine production, and the lysine content was determined inthe culture supernatant.

For this purpose, the clones were first propagated on brain-heart agarplates (Merck, Darmstadt, Germany) at 33° C. for 24 hours. Starting fromthese agar plate cultures, in each case a preculture was inoculated (10ml of medium in a 100 ml Erlenmeyer flask). The medium used for saidpreculture was MM medium. The preculture was incubated on a shaker at33° C. and 240 rpm for 24 hours. From this preculture, a main culturewas inoculated in such a way that the starting OD (660 nm) of said mainculture was 0.1 OD. The MM medium was likewise used for the mainculture.

Medium MM

CSL 5 g/l MOPS 20 g/l Glucose (autoclaved separately) 50 g/l Salts:(NH₄)₂SO₄) 25 g/l KH₂PO₄ 0.1 g/l MgSO₄*7H₂O 1.0 g/l CaCl₂*2H₂O 10 mg/lFeSO₄*7H₂₀ 10 mg/l MnSO₄*H₂O 5.0 mg/l Biotin (sterile-filtered) 0.3 mg/lThiamin * HCl (sterile-filtered) 0.2 mg/l CaCO₃ 25 g/l

CSL (corn steep liquor), MOPS (morpholinopropanesulfonic acid) and thesalt solution were adjusted to pH 7 with aqueous ammonia and autoclaved.Subsequently, the sterile substrate and vitamin solutions and thedry-autoclaved CaCO₃ were added.

Culturing took place in volumes of 10 ml contained in 100 ml Erlenmeyerflasks with baffles. The temperature was 33° C., the number ofrevolutions per minute was 250 rpm and the humidity was 80%.

After 24 hours, the optical density (OD) was determined at a measuringwavelength of 660 nm using a biomek 1000 device (Beckmann InstrumentsGmbH, Munich, Germany). The amount of lysine formed was determined byion exchange chromatography and post-column derivatization withninhydrin detection, using an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany). A mutant distinguished by anincreased lysine formation was referred to as DM1816.

TABLE 1 Lysine-HCl Strain OD (660) (g/l) DM1797 11.7 3.6 DM1816 11.8 3.9

Example 3

Sequencing of the zwf Gene of the DM1816 Mutant

Chromosomal DNA was isolated from the DM1816 clone by the method ofEikmanns et al. (Microbiology 140: 1817-1828 (1994)). A DNA sectioncarrying the zwf gene was amplified with the aid of the polymerase chainreaction. To this end, the following oligonucleotides were used asprimers:

zwf-L1 (SEQ ID NO: 19): 5′ agaagctgac gctgtgttct 3′ zwf-L2 (SEQ ID NO:20): 5′ cattggtgga ctcggtaact 3′

The primers depicted were synthesized by MWG Biotech (Ebersberg,Germany). They enable an approx. 1.95 kb DNA section carrying the zwfgene to be amplified. The zwf-L1 primer binds to the regioncorresponding to positions 59 to 78 of the strand complementary to SEQID NO:3. The zwf-L2 primer binds to the region corresponding topositions 2026 to 2007 of the strand according to SEQ ID NO:3.

The PCR reaction was carried out using the Phusion High Fidelity DNApolymerase (New England Biolabs, Frankfurt, Germany). The reactionmixture was prepared according to the manufacturer's instructions andcontained in a total volume of 50 μl 10 μl of the supplied 5× Phusion HFbuffer, deoxynucleoside triphosphates, in each case in a concentrationof 200 μM, primers in a concentration of 0.5 μM, approximately 50 ng oftemplate DNA and 2 units of Phusion polymerase. The volume was adjustedto 50 μl by adding H₂O.

The PCR mixture was first subjected to an initial denaturation at 98° C.for 30 seconds. This was followed by 35 repeats of a denaturation stepat 98° C. for 20 seconds, a step of binding the primers to the initiallyintroduced DNA at 60° C. for 20 seconds and the extension step ofextending the primers at 72° C. for 60 seconds. After the finalextension step of 5 minutes at 72° C., the PCR mixture was subjected toan agarose gel electrophoresis (0.8% agarose). An approx. 1.85 kb DNAfragment was identified, isolated from the gel and purified using theQIAquick Gel Extraction Kit from Qiagen, (Hilden, Germany).

The nucleotide sequence of the amplified DNA fragment and PCR product,respectively, was determined by Agowa (Berlin, Germany). The sequenceobtained of the coding region of the zwf allele is depicted in SEQ IDNO:9. The amino acid sequence of the protein, established with the aidof the Patentin program, is depicted in SEQ ID NC:10.

The nucleotide sequence of the coding region of the zwf allele of theDM1816 mutant comprises the nucleobase adenine in position 961 (see SEQID NO:5 or 9). The wild type gene (see SEQ ID NO:1) comprises thenucleobase guanine in this position. This guanine-adenine transitionresults in an amino acid substitution of serine for glycine in position321 of the resulting amino acid sequence. This mutation is referred toas zwfG321S hereinbelow. The zwf allele of DM1816 furthermore alsocomprises the nucleotide substitution of thymine with adenine inposition 22 of the nucleotide sequence. This thymine-adeninetransversion results in an amino acid substitution of threonine forserine in position 8 of the resulting amino acid sequence.

Moreover, the zwf allele of DM1816 also comprises another fivenucleotide substitutions which do not result in an amino acidsubstitution (“silent mutations”): a cytosine-thymine transition inposition 138, a cytosine-thymine transition in position 279, athymine-cytosine transition in position 738, a cytosine-thyminetransition in position 777 and a guanine-adenine transition in position906.

EXAMPLE 4

Construction of the Exchange Vector pK18mobsacB_zwfG321S

A part of the coding region, i.e. an “internal fragment” or “internalregion”, of the zwf allele, which carries the mutation zwfG321S, wasamplified with the aid of the polymerase chain reaction. The templateused was the chromosomal DNA obtained in example 3. The followingoligonucleotides were selected as primers for the PCR:

zwf-int1-bam (SEQ ID NO: 23): 5′ ctag-ggatcc-acgtacgcgatgccgcaagt 3′zwf-int2-bam (SEQ ID NO: 24): 5′ ctag-ggatcc-tcaggctgcacgcgaatcac 3′

They were synthesized by MWG Biotech (Ebersberg, Germany) and enable anapprox. 1 kb DNA section of the coding region to be amplified. Thenucleotides 11 to 30 of the zwf-int1-ban primer bind to the regioncorresponding to positions 546 to 565 of the strand complementary to SEQID NO:3. The positions 546 and 565 of SEQ ID NO:3 correspond topositions 239 and 258 in SEQ ID NO:1. The nucleotides 11 to 30 of thezwf-int2-bam primer bind to the region corresponding to positions 1527to 1508 of the strand according to SEQ ID NO:3. The positions 1527 and1508 of SEQ ID NO:3 correspond to positions 1220 and 1201 of SEQ IDNO:1. The primers moreover comprise the sequences for cleavage sites ofthe restriction endonuclease BamHI, which are indicated by underscoringin the nucleotide sequence depicted above.

The PCR reaction was carried out using the Phusion High-Fidelity DNApolymerase (New England Biolabs, Frankfurt, Germany). The reactionmixture was composed as described above. The PCR was carried out asabove, with one exception: the 72° C. extension step in the 35 repeatswas carried out in each case only for 30 seconds.

The approx. 1 kb amplicon was treated with the BamHI restrictionendonuclease and identified by electrophoresis in a 0.8% strengthagarose gel. It was subsequently isolated from the gel and purifiedusing the QIAquick Gel Extraction Kit from Qiagen.

The DNA fragment purified in this way comprises the zwfG321S mutationdescribed and has BamHI-compatible ends (zwfG321S fragment and,respectively, ‘zwf’ in FIG. 1). It was then incorporated into themobilizable pK18mobsacB vector described by Schäfer et al. (Gene, 145,69-73 (1994)), in order to enable an allele or mutation exchange. Tothis end, pK18mobsacB was digested with the BamHI restriction enzyme andthe ends were dephosphorylated by alkaline phosphatase (AlkalinePhosphatase, Boehringer Mannheim, Germany). The thus prepared vector wasmixed with the zwfG321S fragment, and the mixture was treated with theReady-To-Go T4 DNA Ligase Kit (Amersham-Pharmacia, Freiburg, Germany).

The ligation mixture was then used to transform the E. coli strain S17-1(Simon et al., Bio/Technologie 1: 784-791, 1993) (Hanahan, In. DNAcloning. A practical approach. Vol. 1. ILR-Press, Cold Spring Harbor,New York, 1989). Selection for plasmid-harboring cells was carried outby plating the transformation mixture on LB agar (Sambrock et al.,Molecular Cloning: a laboratory manual. 2^(nd) Ed. Cold Spring Harbor,N.Y., 1989) which had been supplemented with 25 mg/l kanamycin.

Plasmid DNA was isolated from a transformant with the aid of the QIAprepSpin Miniprep Kit from Qiagen and checked by restriction cleavage, ineach case once with the enzyme BamHI and once with the enzyme SacI, andsubsequent agarose gel electrophoresis. The plasmid was namedpK18mobsacB_zwfG321S and is depicted in FIG. 1.

EXAMPLE 5

Incorporation of the zwfG321S Mutation into the DM1797 Strain

The pK18mobsacB_zwfG321S vector described in example 4 was transferredby way of conjugation into the C. glutamicum strain DM1797 according tothe protocol by Schäfer et al. (Journal of Microbiology 172: 1663-1666(1990)). The vector cannot self-replicate in DM1797 and remains in thecell only when integrated in the chromosome as a result of arecombination event. The selection of transconjugants, i.e. of cloneswith integrated pK18mobsacB_zwfG321S, was carried out by plating theconjugation mixture on LB agar which had been supplemented with 25 mg/lkanamycin and 50 mg/l nalidixic acid. Kanamycin-resistanttransconjugants were then streaked out on LB agar plates supplementedwith kanamycin (25 mg/l) and incubated at 33° C. for 24 hours. Mutantsin which the plasmid had been excised as a result of a secondrecombination event were selected by culturing the clonesnon-selectively in LB liquid medium for 30 hours, then streaking themout on LB agar which had been supplemented with 10% sucrose andincubating at 33° C. for 24 hours.

Like the pK18mobsacB starting plasmid, the pK18mobsacB_zwfG321S plasmidcomprises, in addition to the kanamycin resistance gene, a copy of thesacB gene coding for Bacillus subtilis levan sucrase. Sucrose-inducibleexpression of the sacB gene results in the formation of levan sucrasewhich catalyzes the synthesis of the product levan which is toxic to C.glutamicum. Therefore, only those clones in which the integratedpK18mobsacB_zwfG321S has been excised as the result of a secondrecombination event grow on sucrose-supplemented LB agar. Depending onthe location of the second recombination event with respect to the siteof mutation, the excision causes the allele exchange or incorporation ofthe mutation, or the original copy remains in the chromosome of thehost.

Subsequently, a clone was looked for in which the desired exchange, i.e.incorporation of the zwfG321S mutation, had taken place. To this end,the sequence of the zwf gene of 10 clones having the phenotype “growthin the presence of sucrose” and “no growth in the presence of kanamycin”was determined. In this way, a clone harboring the zwfG321S mutation wasidentified. This strain was referred to as C. glutamicumDM1797_zwfG321S.

EXAMPLE 6

Comparison of the Performance of the DM1797_zwfG321S Strain with that ofthe DM1797 Starting Strain

The performance test was carried out as described in example 2. TheDM1797_zwfG321S strain displayed, in comparison with DM1797, a markedlyincreased secretion of lysine, similar to DM1816 (see table 1).

What is claimed is:
 1. A process for preparing an L-amino acid,comprising: a) fermenting a coryneform bacterium in a suitable medium toproduce a fermentation broth containing biomass, wherein said coryneformbacterium comprises the polypeptide of SEQ ID NO:2 except that anyproteinogenic amino acid other than glycine is present at position 321and/or any proteinogenic amino acid other than serine is present atposition 8, and wherein said polypeptide has glucose 6-phosphatedehydrogenase activity; and b) collecting, isolating or purifying theL-amino acid being prepared from the fermentation broth and/or saidcoryneform bacterium along with 0 to 100% by weight of said biomass. 2.The process of claim 1, wherein: a) the L-amino acid prepared isselected from the group consisting of : L-aspartic acid, L-asparagine,L-threonine, L-serine, L-glutamic acid, L-glutamine, glycine, L-alanine,L-cysteine, L-valine, L-rnethionine, L-isoleucine, L-leucine,L-tyrosine, L-phenylalanine, L-histidine, L-lysine, L-tryptophan,L-proline and L-arginine. L-homoserine likewise belongs to the L-aminoacids; and b) said coryneform bacterium comprises the polypeptide of SEQID NO:2 except that any proteinogenic amino acid other than glycine ispresent at position 321 and any proteinogenic amino acid other thanserine is present at position
 8. 3. The process of claim 1, wherein theL-amino acid prepared is L-lysine.
 4. The process of claim 1, whereinthe L-amino acid prepared is L-tryptophan.
 5. The process of claim 1,wherein said coryneform bacterium is selected from the group consistingof: Corynebacterium efficiens; Corynebacterium glutamicum;Corynebacterium thermoaminogenes; and Corynebacterium aminogenes.
 6. Theprocess of claim 1, wherein said coryneform bacterium is Corynebacteriumglutamicum.
 7. The process of claim 2, wherein said L-amino acid beingprepared is obtained by collecting and drying the fermentation brothproduced in step a) along with said biomass.
 8. The process of claim 2,wherein said L-amino acid being prepared is purified from thefermentation broth produced in step a).
 9. A process for preparingL-lysine, comprising: a) fermenting a coryneform bacterium in a suitablemedium to produce a fermentation broth containing biomass, wherein saidcoryneform bacterium comprises the polypeptide of SEQ ID NO:2 exceptthat any proteinogenic amino acid other than glycine is present atposition 321 and/or any proteinogenic amino acid other than serine ispresent at position 8, and wherein said polypeptide has glucose6-phosphate dehydrogenase activity; and b) either collecting saidfermentation broth along with said biomass or purifying said L-lysinefrom said fermentation broth or said biomass.
 10. The process of claim9, wherein said fermentation broth is collected along with said biomassand then died.
 11. The process of claim 10, wherein said coryneformbacterium comprises the polypeptide of SEQ ID NO:2 except that anyproteinogenic amino acid other than glycine is present at position 321and any proteinogenic amino acid other than serine is present atposition
 8. 12. The process of claim 11, wherein said coryneformbacterium is Corynebacterium glutamicum.
 13. The process of claim 9,wherein said L-lysine is purified from said fermentation broth.
 14. Theprocess of claim 10, wherein: a) said coryneform bacterium is selectedfrom the group consisting of: Corynebacterium efficiens; Corynebacteriumglutamicum; Corynebacterium thermoaminogenes; and Corynebacteriumaminogenes; and b) said coryneform bacterium comprises the polypeptideof SEQ ID NO:2 except that any proteinogenic amino acid other thanglycine is present at position 321 and any proteinogenic amino acidother than serine is present at position
 8. 15. A process for preparingL-tryptophan, comprising: a) fermenting a coryneform bacterium in asuitable medium to produce a fermentation broth, wherein said coryneformbacterium comprises the polypeptide of SEQ ID NO:2 except that anyproteinogenic amino acid other than glycine is present at position 321and/or any proteinogenic amino acid other than serine is present atposition 8, and wherein said polypeptide has glucose 6-phosphatedehydrogenase activity; and b) either collecting said fermentation brothalong with said biomass or purifying said L-tryptophan from saidfermentation broth or said biomass.
 16. The process of claim 15, whereinsaid fermentation broth is collected along with said biomass and thendied.
 17. The process of claim 16, wherein said coryneform bacteriumcomprises the polypeptide of SEQ ID NO:2 except that any proteinogenicamino acid other than glycine is present at position 321 and anyproteinogenic amino acid other than serine is present at position
 8. 18.The process of claim 17, wherein said coryneform bacterium isCorynebacterium glutamicum.
 19. The process of claim 15, wherein saidL-tryptophan is purified from said fermentation broth.
 20. The processof claim 19, wherein: a) said coryneform bacterium is selected from thegroup consisting of: Corynebacterium efficiens; Corynebacteriumglutamicum; Corynebacterium thermoaminogenes; and Corynebacteriumaminogenes; and b) said coryneform bacterium comprises the polypeptideof SEQ ID NO:2 except that any proteinogenic amino acid other thanglycine is present at position 321 and any proteinogenic amino acidother than serine is present at position 8.